BIOC2201 Learning Outcome Short Responses (Weeks 1-4)
Principles of Molecular Biology (advanced) (University of New South Wales)
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BIOC2201 Learning Outcomes
WEEK 1
Lecture 1: Nucleic Acids I
E[plain how DNA and RNA are linear pol\mers of nucleotides and how each
component of the nucleotide is joined to form this chain of pol\mers (e.g. base to
base; sugar to base; nucleoside to phosphate; nucleotide to nucleotide).
DNA and RNA aUe lineaU Sol\meUV of nXcleoWideV, Zhich aUe conVideUed Whe ³bXilding blockV´ of
boWh nXcleic acidV. NXcleoWideV aUe Whe monomeUV WhaW make XS nXcleic acidV, Whe\ join WogeWheU
Yia ShoVShodieVWeU bondV Wo foUm WheiU Sol\meUV, nXcleic acidV.NXcleoWideV aUe comSoVed of
WhUee comSonenWV, a niWUogenoXV baVe (SXUineV oU S\UimidineV), a VXgaU (UiboVe oU deo[\UiboVe)
and a ShoVShoUic acid (ShoVShaWe). The nXcleic acidV Whe\ foUm aUe deSendenW on Whe
niWUogenoXV baVeV and VXgaU XVed.
Recognise and identif\ from their chemical structure:
a. Ribose and deo[\ribose sugars
RNA iV comSoVed of UiboVe VXgaU, Zhich conWainV a h\dUo[\l gUoXS on Whe 3¶ and 2¶ caUbon.
ComSaUaWiYel\, DNA iV comSoVed of deo[\UiboVe VXgaU, Zhich conWainV a h\dUo[\l gUoXS on Whe
3¶ caUbon, bXW lackV an o[\gen on Whe 2¶ caUbon.
b. Purines and p\rimidines
NiWUogenoXV baVeV can eiWheU be SXUine (adenine and gXanine) oU S\UimidineV (Wh\mine,
c\WoVine and XUacil). One of Whe moVW defining feaWXUeV of SXUineV iV WheiU 2 heWeUoc\clic UingV,
ZheUe Whe coXnWing of caUbonV and niWUogenV iV clockZiVe. In adenine, WheUe iV an amino gUoXS
on Whe 6Wh caUbon, ZhilVW gXanine haV an amino gUoXS on Whe 2nd caUbon and a keWone gUoXS on
Whe 6Wh caUbon. ComSaUaWiYel\, S\UimidineV onl\ haYe 1 heWeUoc\clic Uing, ZheUe Whe coXnWing of
caUbonV and niWUogenV iV anWiclockZiVe. In c\WoVine, WheUe iV a keWone gUoXS on Whe 2nd caUbon
and an amino acid on Whe 4Wh caUbon, ZhilVW in Wh\mine WheUe iV a keWone gUoXS aW SoViWionV 2
and 4 and meWh\l gUoXS aW SoViWion 5 and in XUacil WheUe aUe 2 keWone gUoXSV aW SoViWionV 2 and 4
UeVSecWiYel\ like Wh\mine, bXW lackV a meWh\l gUoXS. WhilVW boWh RNA and DNA aUe comSoVed of
Whe Vame SXUineV, adenine and gXanine, DNA¶V S\UimidineV inclXde Wh\mine and c\WoVine ZhilVW
RNA¶V S\UimidineV inclXde XUacil and c\WoVine. In boWh nXcleic acidV, SXUineV and S\UimidineV Zill
onl\ bind Wo iWV coXnWeUSaUW, ZheUe adenine Zill alZa\V bind Yia h\dUogen bondV Wo Wh\mine foU
DNA and XUacil foU RNA, and gXanine Zill alZa\V bind ZiWh c\WoVine Yia h\dUogen bondV.
c. The nucleosides
The baVe and VXgaU foU Whe nXcleic acid, Zhen joined b\ gl\coVidic bondV, aUe knoZn aV
nXcleoVideV. In SXUineV, Whe SoinW of aWWachmenW of Whe VXgaU iV on Whe 1¶-C aWom Wo Whe N-9
niWUogen of Whe niWUogenoXV baVe. In S\UimidineV, Whe SoinW of aWWachmenW of Whe VXgaU iV of Whe
1¶-C aWom Wo Whe N-1 niWUogen of Whe niWUogenoXV baVe.
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d. Purine and p\rimidine nucleotides and their phosphor\lated derivatives
(AMP, ADP, ATP, etc.)
PXUineV and S\Uimidine nXcleoWideV can be giYen ShoVShoU\laWed deUiYaWiYeV. When naming Whe
nXcleoVide ShoVShoUic acid eVWeUV, Whe nXmbeU of ShoVShaWeV iV eVWeUified Wo Whe 5¶-OH gUoXS of
Whe VXgaU WhaW deWeUmineV iWV name. FoU e[amSle, if Whe baVe iV adenine and iW iV joined Wo a
UiboVe VXgaU Yia gl\coVidic bond, WhaW nXcleoVide iV called adenoVine. If onl\ one ShoVShaWe iV
eVWeUified on Whe 5¶-OH of Whe VXgaU, Whe deUiYaWiYe ZoXld be named adenoVine
5¶-monoShoVShaWe, alVo knoZn aV AMP.
E[plain in detail the bonds/linkages that occur between each of the components
in this chain of pol\mers (e.g. joining: base to base; sugar to base; nucleoside to
phosphate; nucleotide to nucleotide).
In nXcleic acidV, WheUe aUe bondV/linkageV beWZeen baVe Wo baVe, VXgaU Wo baVe and nXcleoVide
Wo ShoVShaWe and nXcleoWide Wo nXcleoWide WhaW aUe made XVing diffeUenW molecXleV. FoU baVe Wo
baVe linkageV, a h\dUogen bond iV XVed, ZheUe SXUineV can onl\ bind Wo WheiU comSlemenWaU\
S\Uimidine baVe and Yice YeUVa. In VXgaU Wo baVe linkage, a gl\coVidic bond iV XVed, ZheUe Whe
SoinW of aWWachmenW iV on Whe C-1¶ of Whe VXgaU and Whe N-9 niWUogen of Whe baVe foU SXUineV and
Whe SoinW of aWWachmenW iV Whe C-1¶ of Whe VXgaU and N-1 niWUogen of Whe baVe in S\UimidineV.
When a baVe and VXgaU bind, iW iV knoZn aV a nXcleoVide. FXUWheUmoUe, foU nXcleoVide Wo
ShoVShaWe linkage, an eVWeU bond iV XVed, ZheUe Whe ShoVShaWe gUoXS iV aWWached Wo Whe 5¶-C
aWom of Whe VXgaU. When a nXcleoVide and ShoVShaWe bind, iW iV knoZn aV a nXcleoWide.
NXcleoWide Wo nXcleoWide linkage occXUV beWZeen Whe 3¶-OH gUoXS of one VXgaU molecXle, and
Whe 5¶-C aWom of anoWheU VXgaU molecXle XVing ShoVShodieVWeU bondV. SeYeUal nXcleoWide Wo
nXcleoWide linkage UeVXlWV in a Sol\nXcleoWide Wo cUeaWe a nXcleic acid.
E[plain the structure of the DNA double heli[ and how it is organised into
chromatin.
A DNA doXble heli[ occXUV Zhen WZo Sol\meUV of DNA aUe joined WogeWheU. The DNA doXble
heli[ conViVWV of WZo Vide chainV WhaW UXn anWiSaUallel Wo one anoWheU. ThiV meanV ZhilVW one
VWUand UXnV in a 5¶ Wo 3¶ diUecWion, Whe oSSoViWe VWUand UXnV a 5¶ Wo 3¶ diUecWion in Whe oSSoViWe
diUecWion. DNA iV V\nWheViVed in Whe 5¶ Wo 3¶ diUecWion, meaning Whe nXcleoWide XniWV aUe onl\
added Wo Whe 3¶ end of Whe nXcleic acid inVWead of Whe 5¶ end. FXUWheUmoUe, Whe WZo DNA VWUandV
aUe noW SeUfecWl\ idenWical Wo one anoWheU dXe Wo Whe VSecificiW\ of WheiU baVe SaiUing inWeUacWion in
Whe cenWUe a[iV of Whe doXble heli[, alWhoXgh Whe\ aUe SUeciVe comSlemenWV Wo one anoWheU. DNA
iV WighWl\ Sacked in Whe nXcleXV in Whe foUm of chUomaWin. The SUoWein iV aVVociaWed ZiWh
chUomaWin, aV iW iV UeVSonVible foU coiling Whe DNA inWo chUomaWin foU oSWimal VWoUage in a cell¶V
nXcleXV.
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Lecture 2: Nucleic Acids II
E[plain the structure of DNA and RNA (mRNA, tRNA, rRNA).
BoWh DNA and RNA aUe nXcleic acidV WhaW conWain baVeV, VXgaUV and ShoVShaWe.
DNA iV comSoVed of Whe niWUogenoXV baVeV, adenoVine, c\WoVine, gXanine and Wh\mine,
ZiWh deo[\UiboVe VXgaU and ShoVShaWe. In DNA, Whe diVWance in baVe SaiUV mXVW alZa\V be
1.08nm. ThiV iV Zh\ SXUine baVeV cannoW bind WogeWheU (diVWance ZoXld be Woo laUge) and WZo
S\UimidineV cannoW bind WogeWheU (diVWance ZoXld be Woo Vmall). The DNA doXble heli[ iV alVo
h\dUoShobic in iWV coUe Wo SUeYenW ZaWeU fUom diVWXUbing Whe h\dUogen bondV ZiWhin, and
h\dUoShilic on iWV VXUface. DNA doXble heliceV alVo conWain majoU and minoU gUooYeV dXe Wo iWV
aV\mmeWU\, ZheUe DNA backboneV aUe cloVeU WogeWheU in minoU gUooYeV and fXUWheU aSaUW in
majoU gUooYeV. AddiWionall\, WheUe aUe WhUee diffeUenW W\SeV of DNA, B-DNA, Z-DNA and H-DNA.
B-DNA iV Whe moVW common foUm of DNA, ZiWh a UighW-handed heli[ WXUn and 10.8 baVe SaiU SeU
WXUn. Z-DNA iV Whe alWeUnaWe lefW-handed foUm of DNA WhaW haV 12 baVe SaiUV SeU WXUn ZhilVW
H-DNA haV WUiSle heliceV foUmed b\ WUinXcleoWide UeSeaWV and iV moVWl\ foXnd in geneWic
diVeaVeV.
ChUomaWin iV
ComSaUaWiYel\, RNA iV comSoVed of Whe niWUogenoXV baVeV, adenoVine, c\WoVine,
gXanine and XUacil, ZiWh UiboVe VXgaU and ShoVShaWe.
E[plain the two specific functions of DNA as the genetic material, and the central
dogma of molecular biolog\.
The cenWUal dogma of molecXlaU biolog\ iV WhaW DNA UeSlicaWeV inWo moUe DNA Yia DNA
UeSlicaWion, and DNA iV XVed Wo cUeaWe RNA Zhich iV When XVed Wo cUeaWe SUoWeinV Yia
WUanVcUiSWion and WUanVlaWion. GeneWic maWeUial mXVW be able Wo UeSlicaWe iWVelf Wo enVXUe Whe
daXghWeU cellV of SaUenWal cellV conWain Whe Vame geneWic maWeUial, hence DNA UeSlicaWion iV
imSeUaWiYe. DXUing DNA UeSlicaWion, comSlemenWaU\ baVe SaiUing, ZheUe adenoVine iV alZa\V
SaiUed ZiWh Wh\mine and gXanine alZa\V SaiUV ZiWh c\WoVine, iV XVed Wo enVXUe an e[acW coS\ of
Whe oUiginal DNA can be made. FXUWheUmoUe, WUanVcUiSWion and WUanVlaWion aUe alVo imSeUaWiYe aV
iW iV Whe cenWUal dogma of molecXlaU biolog\. TUanVcUiSWion iV Whe SUoceVV ZheUe Whe geneWic code
in a VSecific SoUWion of DNA iV WUanVcUibed inWo meVVengeU RNA (mRNA). TUanVlaWion iV Whe
SUoceVV ZheUe mRNA iV WUanVlaWed inWo a SUoWein Yia SUoWein V\nWheViV.
E[plain the basic function for each of the three t\pes of RNA.
The WhUee W\SeV of RNA inclXde meVVengeU RNA (mRNA), WUanVfeU RNA (WRNA), and UiboVomal
RNA (URNA). mRNA onl\ comSoVeV 5% of all RNA, and iV UeVSonVible foU WUanVcUibing geneWic
maWeUial fUom DNA Wo be laWeU XVed Wo code foU SUoWein V\nWheViV. WRNA accoXnWV foU 15%and iV
UeVSonVible foU caUU\ing amino acidV Wo Whe UiboVomeV aV diUecWed b\ Whe comSlemenWaU\
VeTXenceV of Whe codonV in mRNA. URNA iV Whe moVW abXndanW foUm of RNA, accoXnWing foU
80% of all RNA, iW iV UeVSonVible foU caWal\Ving SUoWein V\nWheViV b\ binding onWo boWh mRNA
and WRNA Wo V\nWheViVe SUoWeinV Yia WUanVlaWion.
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E[plain the chemical properties of DNA and RNA with respect to pH, UV
absorbance and temperature, and wh\ DNA is susceptible to breakage.
The chemical SUoSeUWieV of DNA and RNA diffeU deSending on Whe nXcleic acid Zhen e[SoVed Wo
diffeUenW SH, UV abVoUbance and WemSeUaWXUeV.
DNA iV TXiWe VWable aW Whe 5-9 SH Uange, ZhilVW RNA iV TXiWe VWable aW Whe 4-5 SH Uange.
ShoXld Whe SH become Woo acidic oU alkaline foU boWh nXcleic acidV, Whe\ ZoXld XndeUgo
deVWabiliVaWion. FoU boWh nXcleic acidV, a decUeaVe in Whe SH leYel, making acidic condiWion ZiWh a
SH leVV Whan 1, ZoXld lead Wo Whe bUeakage of ShoVShodieVWeU bondV beWZeen Whe nXcleoWideV,
bUeakageV in Whe gl\coVidic bondV in Whe nXcleoVideV and Whe VeSaUaWion of Whe
inWeUmolecXlaU/inWUamolecXlaU h\dUogen bondV beWZeen Whe baVe SaiUV. ThiV ZoXld oYeUall caXVe
bUeakage of Whe nXcleic acid VWUandV and Whe comSleWe h\dUol\ViV of WheiU nXcleoWideV. Weak SH
in Whe 2-4 Uange ZoXld caXVe deSXUinaWion of DNA, ZheUe Whe gl\coVidic bondV linking Whe
VXgaUV Wo Whe SXUine baVeV ZoXld be alWeUed. ThiV ZoXld UeVXlW in Whe UemoYal of SXUineV fUom
Whe VXgaU. High SH, ZiWh a 10-13 Uange, ZoXld caXVe RNA Wo become YeU\ XnVWable.
PhoVShodieVWeU bondV WhaW link RNA nXcleoWideV WogeWheU deWach fUom one anoWheU, cUeaWing
RNA fUagmenWV WhaW UeVXlW in fUee RNA nXcleoWideV. ComSaUaWiYel\, DNA iV UelaWiYel\ VWable in
alkaline condiWionV, hoZeYeU iW iV VXVceSWible Wo alkaline denaWXUaWion, ZheUe Whe doXble
VWUanded DNA VWUXcWXUe iV bUoken inWo Vingle VWUandV.
FXUWheUmoUe, boWh DNA and RNA abVoUb UV lighW aW 260nm. Single VWUanded DNA iV
able Wo abVoUb mXch moUe UV lighW Whan doXble VWUanded DNA aV iW doeV noW haYe VWacking
inWeUacWionV of baVeV. ThiV Shenomenon iV knoZn aV h\SeUchUomic VhifW
AddiWionall\, high WemSeUaWXUeV, VXch aV aUoXnd 90žC, caXVeV doXble-VWUanded DNA Wo
become Vingle VWUanded DNA. The melWing WemSeUaWXUe (Tm) of Whe DNA Zill diffeU deSending on
Whe DNA VeTXence, aV DNA ZiWh moUe gXanine and c\WoVine SaiUingV (GC conWenW) Zill haYe a
higheU affiniW\ Whan DNA ZiWh moUe adenine and Wh\mine SaiUingV (AT conWenW) dXe Wo WheiU
addiWional h\dUogen bond.
E[plain the melting temperature (Tm) of DNA, how it is determined, and how this
value will differ between different DNA sequences.
The melWing WemSeUaWXUe of DNA iV Whe SoinW in WemSeUaWXUe ZheUe 50% of DNA iV doXble
VWUanded, and 50% iV Vingle VWUanded. ThiV YalXe Zill diffeU beWZeen DNA VeTXenceV deSending
on Whe DNA VeTXence'V comSoViWion. DNA ZiWh moUe gXanine and c\WoVine SaiUingV (GC
conWenW) Zill haYe a higheU affiniW\ Whan DNA ZiWh moUe adenine and Wh\mine SaiUingV (AT
conWenW) dXe Wo WheiU addiWional h\dUogen bond.
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WEEK 2
Lecture 3: Nucleic Acids III
E[plain the five steps in a DNA e[traction including the function of reagents
added during different steps.
The fiYe VWeSV in a DNA e[WUacWion inclXde, cell l\ViV, DNA SXUificaWion, DNA SUeciSiWaWion,
ZaVhing Whe DNA SelleW, and Ueh\dUaWing Whe SelleW.
The fiUVW VWeS of DNA e[WUacWion iV Wo l\Ve (i.e. bUeak) Whe cell membUane and nXcleaU
enYeloSe Wo UeleaVe Whe DNA. Anionic deWeUgenWV, called VodiXm dodec\l VXlShaWe (SDS) aUe
XVed in cell l\ViV VolXWionV. SDS diVUXSWV Whe cell membUane aV iW conWainV non-SolaU
h\dUocaUbon molecXleV ZiWh SolaU endV. ThiV enableV Whe SDS Wo bind Wo Whe SolaU and nonSolaU
endV of Whe ShoVSholiSidV occXS\ing Whe cell membUane and bUeak iW aSaUW, caXVing all Whe cell¶V
conWenWV Wo UeleaVe inWo Whe VolXWion.
The Vecond VWeS of DNA e[WUacWion iV Wo SXUif\ Whe VamSle, Wo enVXUe onl\ DNA fUom Whe
cell iV SUeVenW in Whe VolXWion. PXUificaWion can be done XVing WZo meWhodV, Shenol/chloUofoUm
e[WUacWion and SUoWein SUeciSiWaWion. DXUing Shenol/chloUofoUm e[WUacWion, Shenol/chloUofoUm iV
added Wo UemoYe SUoWeinV, liSidV and cell debUiV. Once Whe VolXWion iV cenWUifXgeV, Whe
Shenol/chloUofoUm foUmV a boWWom la\eU conWaining liSidV, an inWeUmediaWe la\eU conWaining
SUoWeinV, and a WoS la\eU of VXSeUnaWanW conWaining DNA. FXUWheUmoUe, SUoWein SUeciSiWaWion can
alVo be XVed foU DNA SXUificaWion. PUoWein SUeciSiWaWion conWainV a high ValW bXffeU, caXVing a
decUeaVe in Whe VolXbiliW\ of Whe SUoWeinV, caXVing Whe SUoWeinV Wo SUeciSiWaWe in Whe VolXWion.
The WhiUd VWeS of DNA e[WUacWion iV DNA SUeciSiWaWion, ZheUe Whe DNA in Whe VolXWion
mXVW SUeciSiWaWe oXW of Whe VolXWion aV in Whe DNA¶V cXUUenW VWaWe, iW iV VWill diVVolYed in Whe
VolXWion WhaW Ze ZoXld ZanW Wo geW Uid of. The objecWiYe of DNA SUeciSiWaWion iV Wo XVe YaUioXV
chemicalV Wo caXVe Whe DNA Wo SUeciSiWaWe fUom Whe VolXWion inWo an inVolXble Volid of DNA WhaW
can be cenWUifXged Wo concenWUaWe Whe DNA inWo a SelleW. DNA SUeciSiWaWion can occXU Yia WZo
meWhodV, iVoSUoSanol (IPA) SUeciSiWaWion and eWhanol SUeciSiWaWion. NXcleic acidV aUe noW VolXble
in iVoSUoSanol, caXVing Whe DNA Wo noW diVVolYe in IPA, and inVWead SUeciSiWaWe fUom Whe VolXWion.
Gl\cogen can alVo acW aV a co-SUeciSiWaWe aV iW Woo iV noW VolXble in IPA, caXVing iW Wo bind onWo
Whe DNA and WUaS iW fXUWheU aV a SUeciSiWaWe. EWhanol can alVo be XVed foU DNA SUeciSiWaWion aV
VodiXm aceWaWe can diVVociaWe inWo SoViWiYel\ chaUged VodiXm ionV and negaWiYel\ chaUged
aceWaWe ionV. The VodiXm ionV neXWUaliVe Whe negaWiYel\ chaUged ShoVShaWe in DNA, making
DNA leVV h\dUoShilic, enabling Whe DNA Wo SUeciSiWaWe Wo foUm a SUeciSiWaWe in Whe VolXWion. FoU
boWh meWhodV, once Whe VolXWion haV XndeUgone cenWUifXgaWion, Whe DNA SUeciSiWaWe Zill foUm inWo
a SelleW and Whe VXSeUnaWanW can be diVcaUded.
The foXUWh VWeS in DNA e[WUacWion iV Wo ZaVh Whe DNA SelleW Wo diVVolYe an\ ValWV WhaW
ma\ be SUeVenW fUom Whe VXSeUnaWanW. The DNA SelleW iV UeVXVSended in 70% eWhanol Wo
diVVolYe an\ ValWV, and Zhen Whe SelleW iV aiU dUied, Whe eWhanol eYenWXall\ eYaSoUaWeV
The final VWeS of DNA e[WUacWion iV Whe Ueh\dUaWion ShaVe. The DNA cannoW be lefW aV a
SelleW in doZnVWUeam e[SeUimenWV aV iW coXld deVVicaWe, hence iW mXVW be diVVolYed in an
aSSUoSUiaWe h\dUaWion VolXWion. The h\dUaWion VolXWion iV deSendenW on ZhaW DNA iV being XVed,
alWhoXgh XVXall\ ZaWeU oU a VXiWable bXffeU can be XVed.
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E[plain how to e[amine the purit\ of \our DNA e[traction using a NanoDrop
spectrophotometer and the A260/A280 ratio.
DNA SXUiW\ can be e[amined XVing a NanoDUoS VSecWUoShoWomeWeU b\ deWeUmining Whe
abVoUbance of a VamSle aW 260nm and 280nm, ZheUe a A260/A280 UaWio iV When XVed Wo
deWeUmine Whe SXUiW\ of Whe DNA VamSle. The A280/A260 UaWio indicaWeV Whe SXUiW\ of DNA oU
RNA VamSleV b\ diYiding Whe UV abVoUbance Ueading aW 260 nm b\ Whe abVoUbance Ueading aW
280 nm. If Whe UaWio iV beWZeen 1.8 Wo 2.0, When Whe NanoDUoS VSecWUoShoWomeWeU indicaWeV WhaW
Whe VamSle iV fUee fUom SUoWein conWaminaWion. If Whe VamSle iV noW ZiWhin WhiV UaWio, When iW ZoXld
eiWheU need Wo be SXUified fXUWheU Wo deconWaminaWe iW, oU a neZ DNA e[WUacWion ZoXld be
SeUfoUmed.
E[plain agarose gel electrophoresis, including how nucleic acid fragments
migrate through the gel based on si]e, shape and charge, and how to estimate
the si]e of bands.
AgaUoVe gel elecWUoShoUeViV iV XVed Wo anal\Ve, idenWif\ and fXUWheU SXUif\ DNA/RNA fUagmenWV
WhaW diffeU in confoUmaWion. agaUoVe gel elecWUoShoUeViV occXUV oYeU WhUee VWeSV, loading of
agaUoVe gel, elecWUoShoUeViV and d\ing. AgaUoVe gel iV loaded onWo Whe DNA/RNA VamSle,
ZheUe a d\e band becomeV YiVible and iV XVed Wo moniWoU Whe SUogUeVVion of Whe VamSle aV Whe
d\e migUaWeV Wo Whe SoViWiYe elecWUode. ElecWUoShoUeViV When occXUV, ZheUe a cXUUenW iV aSSlied,
caXVing Whe negaWiYel\ chaUged nXcleic acid Wo migUaWe WoZaUdV Whe SoViWiYe elecWUode.
DNA/RNA fUagmenWV moYe WhUoXgh Whe gel baVed on WheiU indiYidXal SUoSeUWieV. FoU e[amSle,
bXlkieU fUagmenWV moYe VloZeU WhUoXgh Whe gel SoUeV dXe Wo WheiU Vi]e and VhaSe and Uemain
cloVeU Wo Whe negaWiYe elecWUode, ZhilVW moUe comSacW fUagmenWV moYe moUe eaVil\ WhUoXgh Whe
SoUeV. The band Vi]eV can be eVWimaWed b\ comSaUing Whem ZiWh knoZn band Vi]eV knoZn aV
Whe laddeU VWandaUd. ThiV alVo aVViVWV in enVXUing Whe bandV aUe of Whe coUUecW Vi]e befoUe XVing
Whe VamSle foU fXUWheU e[SeUimenWaWion. The agaUoVe gel iV When finall\ imaged XndeU UV lighW
XVing inWeUcalaWing d\e Wo SUodXce Whe DNA SaWWeUn.
Describe the t\pes of nucleases, and how endonucleases and e[onucleases
cleave nucleic acids.
NXcleaVeV aUe a gUoXS of en]\meV XVed Wo degUade nXcleic acidV ZiWh chemical VSecificiW\ b\
cleaYing Whe ShoVShodieVWeU bondV beWZeen nXcleoWideV. T\SeV of nXcleaVeV inclXde
deo[\UibonXcleaVeV, WhaW can acW on VVDNA, dVDNA oU boWh, and UibonXcleaVeV, Zhich can onl\
acW on RNA. The W\SeV of nXcleaVeV can eiWheU be endonXcleaVeV oU e[onXcleaVeV.
EndonXcleaVeV acW ZiWhin a nXcleic acid VWUand, Whe\ aUe moUe ViWe VSecific, cleaYing Whe nXcleic
acid aW VSecific ViWeV (eg. UeVWUicWion en]\meV). E[onXcleaVeV acW on Whe end of nXcleic acid
VWUandV and UemoYe a Vingle nXcleoWide aW a Wime aW Whe 3¶ oU 5¶ end of Whe DNA molecXle.
Define what a restriction en]\me cleavage frequenc\ is and how it is calculated.
ReVWUicWion en]\meV aUe a W\Se of endonXcleaVe UeVSonVible foU cleaYing VSecific foUeign nXcleic
acid VeTXenceV befoUe Whe\ enWeU Whe cell. The UeVWUicWion en]\me cleaYage fUeTXenc\ iV XVed Wo
calcXlaWe Whe SUobabiliW\ of a VSecific VeTXence occXUUing in a Uandom VeTXence if Whe DNA
VeTXence iV XnknoZn, and Wo WhXV SUedicW hoZ ofWen a UeVWUicWion en]\me Zill cleaYe a Uandom
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VeTXence. The cleaYage fUeTXenc\ iV calcXlaWed b\ finding Whe SUobabiliW\ of a baVe occXUUing aW
a VSecific ViWe (1 in 4) in Whe VeTXence.
Lecture 4: DNA Replication I
List and e[plain the ten main requirements of DNA replication in a living cell.
The Wen main UeTXiUemenWV of DNA UeSlicaWion in a liYing cell inclXde DNA Sol\meUaVe, Wo
V\nWheViVe Whe DNA Wo be XVed on a WemSlaWe DNA VWUand WhaW iV Wo be UeSlicaWed. A SUimeU,
Zhich iV a VhoUW nXcleic acid ZiWh a fUee UeceVVed 3¶-OH Wo enVXUe Whe DNA Sol\meUaVe can
inWeUacW ZiWh Whe SUimeU iV SUacWicall\ Whe VWaUWing block of Whe neZ DNA VWUand. All foXU dNTPV
and magneViXm aUe needed aV bXilding blockV foU DNA, ZiWh Whe µN¶ being eiWheU A, T, C oU G,
ZhilVW magneViXm iV XVed aV a cofacWoU. FXUWheUmoUe, an oUigin of UeSlicaWion iV needed aV a
VWaUWing SoinW of UeSlicaWion, ZhilVW helicaVe and DNA g\UaVe aUe needed Wo XnUaYel Whe DNA
doXble heli[ VWUand in Whe beginning of UeSlicaWion. VVDNA binding SUoWeinV aUe XVed Wo bind onWo
oSen VWUandV Wo keeS Whe VWUandV VeSaUaWed fUom one anoWheU, ZhilVW SUimaVe helSV make RNA
SUimeU WhaW beginV DNA UeSlicaWion and DNA ligaVe helSV heal Whe gaSV lefW in Whe V\nWheViVed
chainV.
Describe the basic reaction that is catal\sed b\ the DNA pol\merase en]\me.
The Sol\meUaVe chain UeacWion iV Whe baVic UeacWion WhaW iV caWal\Ved b\ Whe DNA Sol\meUaVe
en]\me. In WhiV UeacWion, SaUenWal doXble VWUanded DNA iV XVed in addiWion Wo dNTPV Wo cUeaWe a
SUogen\ DNA and inoUganic S\UoShoVShaWe (PPi) b\ DNA Sol\meUaVe. The dNTP iV
incoUSoUaWed inWo Whe DNA Sol\meUaVe chain UeacWion, binding iW onWo iWV comSlemenWaU\ baVe.
DXe Wo Whe dNTP molecXle oUiginall\ conWaining WhUee ShoVShaWeV, bXW onl\ one iV needed foU Whe
DNA¶V VXgaU-ShoVShaWe backbone, Whe WZo lefWoYeU ShoVShaWeV, in Whe foUm of PPi, aUe WhXV
SUodXced. PPi iV neceVVaU\ aV a dUiYing foUce foU Whe UeacWion Wo conWinXe going foUZaUd,
enVXUing Whe DNA Sol\meUaVe addV nXcleoWideV Wo Whe neZ DNA VWUand, UaWheU Whan UemoYing
nXcleoWideV.
List the main factors that contribute to the accurac\ of DNA replication.
The main facWoUV WhaW conWUibXWe Wo Whe accXUac\ of DNA UeSlicaWion inclXde balanced nXcleoWide
leYelV (nXcleoWideV aUe aW UighW concenWUaWionV), Whe Sol\meUaVe mechaniVm, Sol\meUaVe
SUoof-Ueading mechaniVmV (UecogniVe coUUecW baVe SaiUing Yia acWiYe ViWe VhaSe) and DNA
UeSaiU en]\me acWiYiW\ (deWecW miVWakeV/damage in DNA VWUand fUom baVe SaiUing).
E[plain Oka]aki¶s model of µsemi-discontinuous replication¶ for double-stranded
DNA.
Oka]aki¶V model of µVemi-diVconWinXoXV UeSlicaWion¶ foU doXble VWUanded DNA VWaWeV WhaW 2
anWiSaUallel SaUenW VWUandV aUe V\nWheViVed in diffeUenW Za\V, ZiWh one being a leading VWUand
and Whe oWheU being a lagging VWUand. Since DNA Sol\meUaVe onl\ e[WendV DNA in a 5¶ Wo 3¶
diUecWion, V\nWheViVe occXUV conWinXoXVl\ in Whe leading VWUand. HoZeYeU, in Whe lagging VWUand,
V\nWheViV iV diVconWinXoXV. In Oka]aki¶V model, Whe lagging VWUand iV V\nWheViVed in Whe 5¶ Wo 3¶
diUecWion XVing Oka]aki fUagmenWV, ZheUe fUagmenWV of DNA aUe V\nWheViVed XVing DNA
Sol\meUaVe, and aUe laWeU joined WogeWheU XVing DNA ligaVe.
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List and describe the functions of the different en]\matic activities (pol\merase
and e[onuclease) that ma\ be e[hibited b\ different DNA pol\merases.
DiffeUenW en]\maWic acWiYiWieV, VXch aV Sol\meUaVe and e[onXcleaVe aUe e[hibiWed b\ diffeUenW
DNA Sol\meUaVeV Zhen UegXlaWing DNA. Pol\meUaVeV add nXcleoWideV in a 5¶ Wo 3¶ diUecWion Wo
neZ DNA VWUandV. ComSaUaWiYel\, e[onXcleaVeV aUe XVed aV SUoof-Ueading mechaniVmV.
E[onXcleaVeV UemoYe nXcleoWideV fUom neZl\ made DNA VWUandV boWh in 5¶ Wo 3¶ and 3¶ Wo 5¶
diUecWionV if WheUe iV a miVWake in DNA Sol\meUaVe baVe SaiUing oU Wo UemoYe RNA SUimeUV XVed
Wo begin DNA UeSlicaWion.
Lecture 5: DNA Replication II
List the 7 main proteins that are required for DNA replication in bacteria and
describe each of their functions.
The 7 main SUoWeinV UeTXiUed foU DNA UeSlicaWion in bacWeUia inclXde:
1. HelicaVe: an en]\me WhaW beginV XnZinding Whe DNA doXble heli[
2. DNA g\UaVe: an en]\me WhaW aVViVWV helicaVe in XnZinding Whe DNA doXble heli[
3. Single VWUand binding SUoWeinV: VWabiliVe Whe Vingle VWUandV of DNA once VeSaUaWed. DNA
iV moUe YXlneUable Zhen oSened, aV oWheU molecXleV ZoXld bind Wo Whe nXcleoWideV aUe
Whe\ can geW cleaYed b\ oWheU nXcleic acidV. SSB SUoWeinV aVViVW in SUoWecWing Whe VWUandV
b\ WemSoUaUil\ binding Wo Whe VWUandV Wo enVXUe no oWheU molecXle can
4. PUimaVe: V\nWheViVeV Whe RNA SUimeU WhaW iV UeTXiUed b\ DNA Sol\meUaVe Wo VWaUW Whe
V\nWheViV SUoceVV on Whe WemSlaWe
5. DNA Sol\meUaVe III: an en]\me UeVSonVible foU elongaWing Whe neZ DNA VWUand Yia DNA
V\nWheViV. DNA Sol\meUaVe III doeV Whe majoUiW\ of bacWeUial DNA V\nWheViV and iV a
holoen]\me WhaW conWainV 3 comSonenWV:
Ɣ CoUe en]\me: conWainV Whe 5¶ Wo 3¶ Sol\meUaVe and 3¶ Wo 5¶ e[onXcleaVe
(UeVSonVible foU SUoofUeading nXcleoWideV WhaW aUe added Wo a gUoZing DNA
VWUand)
Ɣ ClamS loadeU: haV man\ fXncWionV, one iV Wo VZiWch beWZeen RNA SUimeU
V\nWheViV and DNA V\nWheViV. The clamS loadeU iV onl\ XVed once in Whe leading
VWUand aW Whe beginning of DNA V\nWheViV Wo add an RNA SUimeU. The clamS
loadeU iV XVed mXlWiSle WimeV on Whe lagging VWUand aV Oka]aki fUagmenWV aUe
XVed, meaning WheUe mXVW be UecXUUing VZiWching beWZeen RNA SUimeU V\nWheViV
and DNA V\nWheViV
Ɣ Sliding clamS: UeVSonVible foU holding Whe holoen]\me comSle[ Wo Whe DNA Wo
make Whe DNA YeU\ SUoceVViYe (en]\me can caUU\ oXW iWV caWal\Wic fXncWion foU a
YeU\ long Wime). If Whe Vliding clamS iV UemoYed iW ZoXld make Whe DNA
Sol\meUaVe III holoen]\me VeSaUaWe fUom Whe WemSlaWe DNA moUe fUeTXenWl\
6. DNA Sol\meUaVe I: en]\me UeVSonVible foU UemoYing Whe RNA SUimeU on maWXUe DNA
and filling Whe gaSV ZheUe RNA SUimeUV ZeUe XVing iWV Sol\meUoXV acWiYiW\.
7. DNA ligaVe: cloVeV Whe ShoVShoeVWeU gaS ZheUe Whe RNA SUimeU ZaV UemoYed Wo foUm a
ShoVShodieVWeU bond
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Describe the basic structure and function of the replisome in E. coli.
The UeSliVome in E. coli iV a WeUm XVed Wo deVcUibe Whe UeSlicaWion machineU\ inYolYed in
caWal\Ving Whe V\nWheViV of Whe leading and lagging VWUandV in DNA UeSlicaWion. The UeSliVome
inclXdeV one DNA Sol\meUaVe III en]\me on Whe lagging and leading VWUandV and a looSing
aUUangemenW on Whe lagging VWUand. The looSing aUUangemenW on Whe lagging VWUand enableV Whe
UeSliVome Wo moYe aV a Vingle XniW aV Whe looS WXUnV Whe lagging VWUand aUoXnd Vo WhaW iW goeV
WhUoXgh Whe DNA Sol\meUaVe III in Whe Vame diUecWion in moYemenW aV Whe leading VWUand. All Whe
VWUXcWXUeV in Whe UeSliVome enableV conWinXoXV UeSlicaWion in Whe leading VWUand and a VeUieV of
RNA SUimeU Oka]aki fUagmenWV efficienWl\.
E[plain how leading and lagging DNA strands are s\nthesised
simultaneousl\ via the replisome in E. coli.
The leading and lagging DNA VWUandV aUe V\nWheViVed Yia Whe UeSliVome, conViVWing of a
WemSlaWe DNA VWUand, RNA SUimeUV, Oka]aki fUagmenWV, WZo DNA Sol\meUaVe III en]\meV and a
looS aUUangemenW. One DNA Sol\meUaVe III en]\me V\nWheViVeV Whe leading VWUand, ZhilVW Whe
oWheU V\nWheViVeV lagging VWUand once SUimaVe haV V\nWheViVed Whe RNA SUimeUV XVed Wo begin
DNA V\nWheViV on Whe WemSlaWe VWUandV and Oka]aki fUagmenWV on Whe lagging VWUand. The
UeSliVome conViVWV of a looSing aUUangemenW on Whe lagging VWUand. The looSing aUUangemenW
UeYeUVeV Whe lagging VWUand Vo WhaW iW iV V\nWheViVed in Whe Vame diUecWion of moYemenW aV Whe
leading VWUand. ThiV alloZV Whe UeSliVome Wo moYe aV a Vingle XniW in Whe diUecWion of Whe
adYancing UeSlicaWion foUk and enVXUe Whe leading and lagging VWUandV aUe V\nWheViVed
VimXlWaneoXVl\.
E[plain the process of the termination of DNA replication in E. coli.
The SUoceVV of WeUminaWion of DNA UeSlicaWion in E.coli occXUV XVing a UeSlicaWion WeUminXV
Uegion. The UeSlicaWion WeUminXV Uegion conViVWV of Wen WeUminaWoU ViWeV WhaW acW aV one Za\
YalYeV, SUeYenWing Whe UeSlicaWion machineU\ (i.e. Whe UeSliVome) fUom back WUacking once Whe\
WUaYel WhUoXgh Whe WeUminXV Uegion. Once boWh UeSlicaWion machineUieV aUUiYe aW Whe WeUminXV
Uegion, WheUe iV a binding of Whe VSecial SUoWein, TXV SUoWein, WhaW linkV Whe neZ VWUandV WhaW haYe
been cUeaWed Wo enVXUe Whe DNA UeSlicaWion SUoceVV endV ZiWh a doXble VWUanded ciUcXlaU
chUomoVome.
Describe the main differences between DNA replication in bacteria and
eukar\otes.
The main diffeUence beWZeen DNA UeSlicaWion in bacWeUia and eXkaU\oWeV iV WhaW Whe eXkaU\oWic
V\VWem iV faU moUe comSle[ dXe Wo Whe amoXnW of DNA WhaW mXVW be UeSlicaWed, and Whe nXmbeU
of SUoWeinV UeTXiUed foU eXkaU\oWic VXUYiYal. In eXkaU\oWic DNA UeSlicaWion, diffeUenW modeV of
UeSlicaWion can occXU. FoU e[amSle, nXcleaU DNA UeSlicaWion can occXU in addiWion Wo
miWochondUia and chloUoSlaVW DNA UeSlicaWion. EXkaU\oWeV alVo XVe VeYeUal diffeUenW DNA
Sol\meUaVeV, ZiWh each conWaining diffeUenW and XniTXe SUoSeUWieV. FXUWheUmoUe, eXkaU\oWic
DNA iV alVo UeSlicaWed fUom mXlWiSle oUiginV, Xnlike bacWeUial DNA ZheUe iW iV onl\ UeSlicaWed fUom
a Vingle oUigin on a ciUcXlaU DNA VWUand. DXe Wo eXkaU\oWic DNA being lineaU and anWiSaUallel, Whe
DNA haV endV and Whe DNA machineU\ Wo WUaYel in oSSoViWe diUecWionV. AV Whe UeSlicaWion
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machineU\ WUaYelV in Whe oSSoViWe diUecWionV, WhiV caVie UeSlicaWion µbXbbleV¶ Wo foUm and incUeaVe
in Vi]e XnWil eYenWXall\ all Whe UeSlicaWion foUkV meeW, UeVXlWing in WZo conWinXoXV VWUandV.
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WEEK 3
Lecture 6: Pol\merase Chain Reaction
E[plain wh\ PCR is s\nthetic DNA replication.
PCR iV a V\nWheWic foUm of DNA UeSlicaWion aV iW caXVeV Whe Vame oXWcome aV DNA UeSlicaWion,
bXW iW occXUV in YiWUo (i.e. in an aUWificial enYiUonmenW) and iW amSlifieV WaUgeWed DNA VeTXenceV.
PCR iV alVo V\nWheWic aV iW doeV noW UeTXiUe all Whe comSonenWV of in YiYo DNA UeSlicaWion,
UeTXiUing onWo foXU oXW of Whe 10 comSonenWV in DNA UeSlicaWion. PCR doeV noW UeTXiUe an oUigin
of UeSlicaWion, aV SUimeUV aUe XVed aV Whe VWaUWing SoinWV foU V\nWheWic UeSlicaWion. FXUWheUmoUe,
en]\meV VXch aV helicaVe, DNA g\UaVe and VVDNA DNA binding SUoWeinV aUe XnneceVVaU\ aV
PCR XVeV heaW Wo VeSaUaWe Whe dVDNA WemSlaWe, ZhilVW SUimaVe iV XnneceVVaU\ aV chemicall\
V\nWheViVed DNA SUimeUV aUe XVed Wo define Whe VWaUWing SoinW foU DNA V\nWheViV. DNA ligaVe iV
alVo noW needed aV no Oka]aki fUagmenWV aUe needed Wo be joined WogeWheU in PCR.
List the 5 components required for PCR and e[plain the purpose of each
component.
The fiYe comSonenWV UeTXiUed foU PCR inclXde:
Ɣ DNA Sol\meUaVe: V\nWheViVeV Whe comSlemenWaU\ dNTPV on a WemSlaWe DNA VWUand WhaW
iV Wo be UeSlicaWed
Ɣ TemSlaWe DNA VWUand: fXncWionV aV an oUigin of UeSlicaWion
Ɣ PUimeU: a VhoUW nXcleic acid SUacWicall\ XVed aV Whe VWaUWing block of a neZ DNA VWUand.
ConWainV a fUee 3¶ h\dUo[\l gUoXS iV needed Wo enVXUe Whe DNA Sol\meUaVe can inWeUacW
ZiWh Whe SUimeU
Ɣ All foXU dNTPV: Whe dNTPS (A, T, C and G) aUe XVed aV bXilding blockV foU DNA
Ɣ Mg2+: cofacWoU UeTXiUed foU DNA Sol\meUaVe Wo fXncWion SUoSeUl\
E[plain the PCR technique, including: the three stages of the PCR c\cle; wh\ the
technique is called the pol\merase chain reaction; and thermal c\cling.
The PCR WechniTXe inYolYeV WhUee oYeUall VWageV of Whe PCR c\cle, denaWXUing, annealing and
elongaWion.
DXUing Whe denaWXUing VWage, Whe dVDNA iV VeSaUaWed inWo WZo VVDNA. ThiV iV neceVVaU\
becaXVe DNA Sol\meUaVe iV Xnable Wo V\nWheViVe a neZ DNA VWUand fUom dVDNA, aV iW UeTXiUeV
a VVDNA Wo Uead off. DenaWXUing iV achieYed b\ heaWing Whe machine Wo UoXghl\ 96žC Wo bUeak
Whe h\dUogen bondV beWZeen Whe nXcleoWide baVe SaiUV. Once Whe baVe SaiUV aUe bUoken aSaUW,
Whe WZo DNA VWUandV Zill VeSaUaWe, foUming indiYidXal Vingle VWUandV of DNA.
DXUing Whe annealing VWage, SUimeUV aUe added Wo Whe VVDNA Wo SUeYenW Whe DNA VWUandV
fUom Uebinding Wo one anoWheU Zhen Whe PCR machine¶V WemSeUaWXUe iV loZeUed. The
WemSeUaWXUe of Whe PCR machine iV UedXced Wo alloZ annealing Wo 48-72žC. The WemSeUaWXUe iV
deSendenW on Whe VeTXence and lengWh of Whe SUimeUV. Annealing Zill caXVe Whe VWUandV Wo noW
Uebind aV Whe SUimeUV added Wo Whe UeacWion aUe in VXch e[ceVV and aUe mXch VhoUWeU in lengWh
comSaUed Wo Whe oUiginal DNA VWUand WemSlaWe. ThiV caXVeV Whe SUimeUV Wo anneal mXch moUe
TXickl\ Whan Whe oUiginal DNA WemSlaWeV.
The final VWage of Whe PCR c\cle iV Whe elongaWion VWage, ZheUe Whe DNA Sol\meUaVe Zill
e[Wend Whe boXnd SUimeUV in Whe 5¶ Wo 3¶ diUecWion b\ adding dNTPV. The DNA Sol\meUaVe, ZiWh
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Whe helS of magneViXm aV a cofacWoU, e[WendV Whe boXnd SUimeUV Wo each Vingle-VWUanded
WemSlaWe DNA VWUand, UeVXlWing in WZo dVDNA molecXleV. DXUing Whe elongaWion VWage, Whe PCR
machine¶V WemSeUaWXUe iV deSendenW on Whe W\Se of DNA Sol\meUaVe XVed. FoU e[amSle, foU TaT
DNA Sol\meUaVe, Whe oSWimal WemSeUaWXUe ZoXld be 72žC. All WhUee VWageV aUe UeSeaWed VeYeUal
WimeV Wo an e[WenW ZheUe Whe WaUgeWed DNA iV eYenWXall\ iVolaWed and UeSlicaWed in e[ceVV Wo be
anal\Ved.
FXUWheUmoUe, WhiV WechniTXe iV knoZn aV Whe Sol\meUaVe chain UeacWion aV iW XVeV a
WheUmoVWable DNA Sol\meUaVe Wo cUeaWe a chain UeacWion of DNA UeSlicaWion, ZheUe Zhen one
UeacWion SUogUeVVeV, cUeaWing PCR SUodXcWV (amSliconV), iW Zill VeW off anoWheU VeW of VimilaU
UeacWionV Wo fXUWheU cUeaWe neZ coSieV of amSliconV. PCR iV alVo knoZn aV WheUmal c\cling aV
Whe PCR machine iV able Wo UegXlaWe and change Whe enYiUonmenW in Zhich Whe DNA iV SUeVenW
WemSeUaWXUe foU each VWeS in PCR.
E[plain wh\ electrophoresis is used in conjunction with PCR to identif\ PCR
products.
AgaUoVe gel elecWUoShoUeViV iV XVed in conjXncWion ZiWh PCR Wo idenWif\ PCR SUodXcWV aV iW iV
XVed Wo anal\Ve Whe PCR SUodXcWV. ElecWUoShoUeViV iV XVed Wo deWeUmine ZheWheU an
amSlificaWion of Whe WaUgeWed VeTXence of DNA haV occXUUed. AgaUoVe gel can be XVed Wo
deWeUmine if Whe PCR SUodXcWV V\nWheViVed aUe Whe e[SecWed Vi]e b\ cUeaWing a DNA laddeU.
WheWheU Whe DNA laddeU iV Whe e[SecWed Vi]e deWeUmineV ZheWheU oU noW Whe WaUgeWed DNA haV
been VeTXenced. FXUWheUmoUe, agaUoVe gel can alVo check Wo Vee if mXlWiSle bandV aUe SUeVenW
ZiWhin Whe DNA VamSle, Zhich ma\ be dXe Wo Whe non-VSecific binding of Whe PCR SUimeUV. If WhiV
ZeUe Wo occXU, iW iV an indicaWion WhaW Whe PCR WemSeUaWXUe VeWWingV ma\ need Wo be UeadjXVWed
oU Whe PCR SUimeUV ma\ need Wo be comSleWel\ UedeVigned.
E[plain which PCR techniques are used in the anal\sis of different nucleic acids.
SeYeUal PCR WechniTXeV aUe XVed Wo anal\Ve diffeUenW nXcleic acidV, VXch aV PCR, UeYeUVe
WUanVcUiSWion PCR and TXanWiWaWiYe PCR. PCR iV XVed Wo amSlif\ doXble-VWUanded DNA, UeYeUVe
WUanVcUiSWion PCR iV XVed Wo anal\Ve mRNA molecXleV b\ XVing iWV comSlemenWaU\ DNA, ZhilVW
TXanWiWaWiYe PCR iV XVed Wo TXanWif\ Whe amoXnW of PCR SUodXcW WhaW iV geneUaWed afWeU each
PCR c\cle. QXanWiWaWiYe SCR iV moVW commonl\ XVed foU ciUcXmVWanceV like COVID WeVWing.
Lecture 7: Transcription
Describe the general structure and mechanism of RNA pol\merases, as the\
compare to DNA pol\merases.
RNA Sol\meUaVeV haYe VomeZhaW VimilaU feaWXUeV Wo DNA Sol\meUaVeV, e[ceSW Whe\ aUe
SUimaUil\ UeVSonVible foU Whe V\nWheViV of SUoWeinV. SimilaU Wo DNA Sol\meUaVeV, RNA
Sol\meUaVeV XVe nXcleoVide 5¶-WUiShoVShaWeV aV SUecXUVoUV Wo SUoWein WUanVcUiSWion and aUe
XVed Wo caWal\Ve Whe ShoVShodieVWeU bondV beWZeen nXcleoWideV foUmed b\ comSlemenWaU\
baVe SaiUing. Like DNA Sol\meUaVeV, RNA Sol\meUaVeV alVo UeTXiUe DNA aV a WemSlaWe Wo
SUodXce Whe comSlemenWaU\ WaUgeW VWUand, and gUoZ Whe neZ nXcleic acid chain fUom Whe 5¶ Wo 3¶
diUecWion on Whe WemSlaWe DNA. Unlike DNA Sol\meUaVeV, RNA Sol\meUaVeV XVe UibonXcleoVide
5¶-WUiShoVShaWe, UaWheU Whan deo[\UibonXcleoVide 5¶-WUiShoVShaWe. FXUWheUmoUe, RNA
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Sol\meUaVeV do noW UeTXiUe a SUimeU Wo iniWiaWe WUanVcUiSWion, onl\ SUodXce a Vingle VWUand of
RNA aV onl\ one VWUand of DNA iV XVed aV a WemSlaWe VWUand, and onl\ WUanVcUibe VhoUW
VWUeWcheV of DNA, ZhilVW DNA UeSlicaWion UeSlicaWeV Whe enWiUe genome.
Describe the basic structure and function of the E. coli RNA pol\merase.
E. coli RNA Sol\meUaVe iV Whe onl\ RNA Sol\meUaVe XVed Wo WUanVcUibe all of a cell¶V RNA and iV
faU leVV comSle[ Whan DNA Sol\meUaVe. E. coli RNA Sol\meUaVe conViVWV of a coUe WeWUameU of
VXbXniWV, and a Vigma VXbXniW. The coUe WeWUameU conViVWV of a beWa-SUime VXbXniW, beWa VXbXniW
and WZo alSha VXbXniWV WhaW aUe all UeVSonVible foU binding Whe RNA Sol\meUaVe Wo Whe DNA
WemSlaWe and ShoVShodieVWeU bond foUmaWion. Sigma VXbXniWV aUe onl\ aVVociaWed ZiWh RNA
Sol\meUaVe Wo helS VWaUW WUanVcUiSWion iniWiaWion. DiffeUenW Vigma VXbXniWV fXncWion b\ UecogniVing
diffeUenW SUomoWeUV. A VSecific Vigma-VXbXniW iV able Wo UecogniVe a VSecific SUomoWeU ViWe on an
oSeUon Wo bind Whe RNA Sol\meUaVe and iniWiaWe WUanVcUiSWion. FXUWheUmoUe, like DNA
Sol\meUaVeV, magneViXm iV alVo a cofacWoU needed foU RNA Sol\meUaVe Wo fXncWion.
List and describe the four main steps that occur in transcription.
The foXU main VWeSV WhaW occXU in WUanVcUiSWion inclXde RNA Sol\meUaVe binding, WUanVcUiSWion
iniWiaWion, elongaWion and WeUminaWion.
RNA V\nWheViV beginV aW VSecific ViWeV on DNA, knoZn aV SUomoWeUV. Onl\ one DNA
VWUand iV XVed aV a WemSlaWe foU RNA V\nWheViV, WhiV VWUand iV knoZn aV Whe anWiVenVe VWUand aV
alWhoXgh iW iV being XVed, iW iV acWXall\ iWV comSlemenWaU\ VWUand WhaW iV being coSied foU RNA
V\nWheViV. ThiV meanV Whe VWUand oSSoViWe Wo Whe anWiVenVe VWUand iV knoZn aV Whe VenVe VWUand,
aV WhiV iV Whe VWUand WhaW iV acWXall\ being coSied. In bacWeUia, geneV aUe ofWen aUUanged in
geneWic XniWV knoZn aV oSeUonV. OSeUonV conWain VSecific WUanVcUiSWion iniWiaWion ViWeV knoZn aV
SUomoWeUV. PUomoWeUV aUe UegionV WhaW aUe UecogniVed b\ coUUeVSonding Vigma-VXbXniWV locaWed
on Whe RNA Sol\meUaVe. Once Whe Vigma-facWoU UecogniVeV iWV coUUeVSonding SUomoWeU, Whe RNA
Zill When be diUecWed Wo iniWiaWe WUanVcUiSWion in a ceUWain locaWion on Whe oSeUon Wo code a VSecific
gene.
TUanVcUiSWion iniWiaWion occXUV once an RNA Sol\meUaVe haV VXcceVVfXll\ boXnd Wo Whe
WaUgeWed SUomoWeU VeTXence on an oSeUon. DXUing WUanVcUiSWion iniWiaWion, Whe RNA Sol\meUaVe
holoen]\me bindV looVel\ Wo non-SUomoWeU DNA Wo alloZ Whe holoen]\me Wo moYe along Whe
DNA XnWil iW deWecWV iWV Vigma-VXbXniW¶V coUUeVSonding SUomoWeU. The RNA Sol\meUaVe Zill When
WighWl\ bind Wo Whe SUomoWeU and elongaWe Whe RNA VWUand fUom Whe 5¶ Wo 3¶ diUecWion, ZhilVW Whe
Vigma-VXbXniW Zill diVVociaWe fUom Whe en]\me.
In RNA WUanVcUiSWion, UaWheU Whan XVing XnZinding en]\meV VXch aV helicaVe and g\UaVe,
RNA Sol\meUaVe binding iV able Wo XnZind Whe DNA enoXgh Wo WUanVcUibe. AV Whe RNA
Sol\meUaVe adYanceV fUom 5¶ Wo 3¶ along Whe DNA, Whe DNA Zill XnZind ahead of Whe RNA¶V 3¶
end, and UeZind dXe Wo baVe SaiUing behind Whe RNA¶V 5¶ end, foUming a WUanVcUiSWion bXbble.
ThiV enableV RNA Sol\meUaVe Wo efficienWl\ WUanVcUibe WaUgeWed DNA, gUoZing Whe RNA chain Yia
elongaWion.
The final VWage of RNA WUanVcUiSWion inYolYeV Whe WeUminaWion of WUanVcUiSWion, ZheUe Whe
WUanVcUiSWion SUoceVV iV inacWiYaWed Yia a WeUminaWion Vignal. The WeUminaWion Vignal in bacWeUial
geneV inclXdeV a UeSeaWed gXanine and c\WoVine Uich VeTXence folloZed b\ foXU adenineV. The
SXUSoVe of WhiV Vignal iV Wo cUeaWe a VWem looS ³haiUSin´ VWUXcWXUe XVing an inYeUWed GC-Uich
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VeTXence WhaW iV eYenWXall\ diVconnecWed b\ Whe VeYeUal comSlemenWaU\ XUacil UeVidXeV. The
GC-Uich VeTXence cUeaWeV a bXlk\, h\dUogen Uich VWUXcWXUe WhaW Whe RNA Sol\meUaVe iV able Wo
bind Wo. HoZeYeU, once Whe RNA Sol\meUaVe adYanceV Wo Whe foXU XUacil molecXleV WhaW foUm
ZeakeU h\dUogen bondV, iW Zill abUXSWl\ diVVociaWe fUom Whe DNA WemSlaWe, SUeYenWing
WUanVcUiSWion fUom conWinXing.
Describe the main differences between transcription in bacteria and eukar\otes.
The main diffeUence beWZeen WUanVcUiSWion in bacWeUia and eXkaU\oWeV iV WhaW eXkaU\oWic
WUanVcUiSWion iV faU moUe comSle[ Whan bacWeUial WUanVcUiSWion. Unlike eXkaU\oWeV, bacWeUial cellV
do noW conWain a nXcleXV, meaning Whe WUanVcUiSWion and WUanVlaWion SUoceVVeV can occXU
VimXlWaneoXVl\. ThiV cannoW occXU in eXkaU\oWic cellV aV Whe\ conWain VeYeUal oUganelleV,
inclXding a nXcleXV, meaning WUanVcUiSWion and WUanVlaWion mXVW occXU aW diffeUenW WimeV. In
eXkaU\oWic cellV, WUanVcUiSWion Zill W\Sicall\ occXU in Whe nXcleXV of Whe cell, ZheUe Whe mRNA
mXVW fiUVW be WUanVSoUWed Wo Whe c\WoSlaVm Wo aWWach Wo a UiboVome, ZheUe WUanVlaWion Zill occXU.
FXUWheUmoUe, eXkaU\oWic WUanVcUiSWion UeTXiUeV mXlWiSle foUmV of RNA Sol\meUaVe aV Zell aV
comSlicaWed conWUol VeTXenceV Wo fXncWion. ThiV makeV Whe eXkaU\oWic WUanVcUiSWion machineU\
moUe comSlicaWed Whan bacWeUial. Unlike bacWeUia, eXkaU\oWeV aUe mXlWicellXlaU, meaning diffeUenW
cellV ma\ fXncWion foU diffeUenW aVSecWV of Whe eXkaU\oWeV, and WheUefoUe ZoXld UeTXiUe diffeUenW
SUoWeinV and fXncWional RNA Wo be made deSending on WheiU fXncWion. TheUefoUe, diffeUenW RNA
Sol\meUaVeV and conWUol VeTXenceV aUe neceVVaU\ foU diffeUenW cellV Wo enVXUe VSecific SUoWeinV
and fXncWional RNA iV V\nWheViVed and UegXlaWed.
List the three main eukar\otic RNA pol\merases, describe their cellular location
and the RNA species that the\ s\nthesise.
The WhUee main eXkaU\oWic RNA Sol\meUaVeV inclXde RNA Sol\meUaVe I, RNA Sol\meUaVe II
and RNA Sol\meUaVe III. RNA Sol\meUaVe I iV locaWed in Whe nXcleoli of Whe cell and iV
UeVSonVible foU V\nWheViVing moVW of Whe URNA SUecXUVoUV. RNA Sol\meUaVe I iV VSecificall\
locaWed in Whe nXcleoli aV URNA molecXleV aUe Whe W\Se of RNA WhaW iV aVVociaWed ZiWh
UiboVomeV. RNA Sol\meUaVe II iV locaWed in Whe nXcleoSlaVm of Whe cell, and iV UeVSonVible foU
V\nWheViVing mRNA, Zhich iV V\nWheViVed in Whe nXcleXV in eXkaU\oWeV. FXUWheUmoUe, RNA
Sol\meUaVe III iV alVo locaWed in Whe nXcleoSlaVm of Whe cell, and iV UeVSonVible foU Whe V\nWheViV
of 5V URNA SUecXUVoUV, and WRNA.
List and describe the promoter features and other DNA elements involved in the
regulation of eukar\otic transcription.
In eXkaU\oWeV, Whe SUomoWeU feaWXUeV and oWheU DNA elemenWV inYolYed in Whe UegXlaWion of DNA
WUanVcUiSWion aUe moUe comSle[ and diYeUVe Whan bacWeUial WUanVcUiSWion UegXlaWion.
Unlike bacWeUial cellV, Whe RNA Sol\meUaVe comSle[ in eXkaU\oWeV doeV noW conWain a
UemoYable Vigma facWoU WhaW findV a VSecific SUomoWeU Uegion Wo iniWial WUanVcUiSWion. EXkaU\oWic
cellV inVWead conWain a nXmbeU of acceVVoU\ SUoWeinV knoZn, VXch aV WUanVcUiSWion facWoUV, WhaW
idenWif\ Whe SUomoWeUV ZiWhin Whe DNA VeTXence and UecUXiW Whe aSSUoSUiaWe RNA Sol\meUaVe Wo
Whe WUanVcUiSWion VWaUW ViWe.
The WhUee main eXkaU\oWic RNA Sol\meUaVeV UecogniVe diffeUenW W\SeV of SUomoWeUV Wo
enVXUe Whe diffeUenW W\SeV of RNA aUe V\nWheViVed ZheUe needed. The WhUee main coUe
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SUomoWeUV SUeVenW in eXkaU\oWic cellV WhaW aVViVW in Whe binding of RNA Sol\meUaVe II Wo DNA
inclXde Whe TATA bo[, GC bo[, and Whe CAAT bo[. The TATA bo[ iV locaWed aSSUo[imaWel\ 25-30
bS XSVWUeam fUom Whe iniWiaWion ViWe in Whe SUomoWeU Uegion. The TATA bo[ coUe SUomoWeU in Whe
DNA deWeUmineV Whe e[acW VWaUW ViWe in moVW SUomoWeUV b\ binding onWo Whe TATA binding SUoWein
WhaW iV a VXbXniW Wo WUanVcUiSWion facWoU II on Whe RNA Sol\meUaVe. The GC bo[ and CAAT bo[
coUe SUomoWeUV fXncWion b\ binding Wo Whe VSecificiW\ facWoU I and CAAT bo[ WUanVcUiSWion facWoU
UeVSecWiYel\ Wo bind Whe aSSUoSUiaWe RNA Sol\meUaVe Wo iWV WaUgeWed DNA VeTXence.
FXUWheUmoUe, oWheU UegXlaWoU\ elemenWV of RNA Sol\meUaVe II, VXch aV WUanVcUiSWion
UegXlaWoU\ elemenWV, fXncWion Wo deWeUmine Whe efficienc\ and fUeTXenc\ of WUanVcUiSWion.
TUanVcUiSWion elemenWV VXch aV enhanceUV, VilenceUV and UeVSonVe elemenWV all fXncWion Wo
UegXlaWe WUanVcUiSWion UaWe and occXUUence. EnhanceUV aUe UeVSonVible foU incUeaVing
WUanVcUiSWion UaWe, VilenceUV decUeaVe WUanVcUiSWion UaWe and UeVSonVe elemenWV WaUgeW
VeTXenceV foU Vignalling molecXleV Wo bind Wo. RegXlaWoU\ SUoWeinV, VXch aV acWiYaWoUV oU
UeSUeVVoUV, can bind Wo WheVe elemenWV Wo eiWheU acWiYaWe oU inacWiYaWe WUanVcUiSWion of ceUWain
geneV deSending on Whe cell¶V need.
List and describe the three main post-transcriptional modifications of eukar\otic
primar\ RNA transcripts.
The WhUee main SoVW-WUanVcUiSWional modificaWionV of eXkaU\oWic SUimaU\ RNA WUanVcUiSWV inclXde
5¶ caSSing, 3¶ Sol\aden\laWion and VSlicing.
DXUing 5¶ caSSing, a 7-meWh\lgXanoVine UeVidXe iV added Wo Whe 5¶ end of Whe mRNA
molecXle Wo aVViVW in idenWif\ing Whe eXkaU\oWic WUanVlaWion VWaUW ViWe, and SUoWecW Whe
Sol\nXcleoWide fUom degUadaWion. EXkaU\oWic UiboVomeV aUe able Wo idenWif\ Whe 5¶ caSSing and
deWecW Whe end of Whe RNA molecXle ZheUe iW can bind and iniWiaWe WUanVlaWion in Whe 5¶ Wo 3;
diUecWion, decoding Whe mRNA inWo a SUoWein. RNA molecXleV aUe W\Sicall\ TXiWe YXlneUable, bXW
Whe 5¶ caS iV able Wo SUoWecW Whe endV of Whe mRNA fUom degUadaWion b\ 5¶-e[onXcleaVe.
3¶ Sol\aden\laWion occXUV Zhen a laUge amoXnW of adenineV aUe added Wo Whe 3¶ end of
Whe mRNA, making iW eaVieU foU eXkaU\oWic UiboVomeV Wo deWeUmine Zhich end of Whe mRNA iW
can bind Wo. 3¶ Sol\aden\laWion occXUV Zhen a cleaYage Vignal aW Whe end of Whe mRNA molecXle
iV cleaYed b\ VSecific endonXcleaVeV, cUeaWing a Vignal Wo add adenine nXcleoWideV XVing ATP.
The Sol\aden\laWed Wail alVo SUoWecWV Whe mRNA fUom 3¶-e[onXcleaVeV and fXncWionV aV a
handle foU Whe SUoWeinV Wo deliYeU mRNA Wo UiboVomeV.
SSlicing iV Whe modificaWion ZheUe Whe mRNA iV SUoceVVed and Whe non-e[SUeVVed
(inWUon) VeTXenceV aUe cXW, and Whe e[SUeVVed (e[on) VeTXenceV aUe joined WogeWheU. SSlicing
UeVXlWV in a mXch VmalleU, maWXUe mRNA molecXle WhaW onl\ conWainV Whe RNA VeTXenceV
needed foU SUoWein WUanVlaWion.
Lecture 8: Translation I
Describe the wa\ in which the genetic code consists of triplet codons that are
read sequentiall\ b\ the ribosome.
TUiSleW codonV ZiWhin a geneWic code aUe VeTXenWiall\ Uead b\ Whe UiboVome aV each codon
conWainV Whe geneWic VeTXence foU a VSecific amino acid. TheUe aUe 64 SoVVible combinaWionV of
geneWic VeTXenceV ZiWhin codonV, Zhich iV moUe Whan enoXgh Wo code foU all 20 amino acidV.
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DXe Wo WheUe being moUe codon VeTXenceV Whan WheUe aUe amino acidV, Vome amino acidV Zill
haYe moUe Whan codon VeTXence, making Whem highl\ degeneUaWe. The codon iV Uead b\ Whe
UiboVome V\VWemaWicall\, ZheUe Whe UiboVome Zill moYe fUom Whe WhUee baVeV of one codon Wo Whe
ne[W WhUee baVeV of Whe VXbVeTXenW codon. FXUWheUmoUe, WheUe aUe VSecific VeTXenceV Wo Vignal
Zhen each codon VWaUWV and VWoSV, knoZn aV Whe Ueading fUame. A mRNA ma\ haYe XS Wo WhUee
diffeUenW Ueading fUameV deSending on ZheUe Whe UiboVome idenWifieV Whe mRNA¶V WUanVlaWion
ViWe. FoU all WhUee diffeUenW Ueading fUameV, diffeUenW codonV aUe foUmed, meaning diffeUenW amino
acidV can be coded fUom Whe Vame mRNA VWUand.
E[plain how each codon represents a specific amino acid or a stop signal in the
µstandard¶ genetic code.
Each codon can UeSUeVenW a VSecific amino acid oU VWoS Vignal in Whe µVWandaUd¶ geneWic code aV
moVW amino acidV aUe highl\ degeneUaWe. Highl\ degeneUaWe amino acidV can be coded b\ moUe
Whan one mRNA codon. In Whe VWandaUd geneWic code, Whe aUUangemenW of codeV iV alVo
non-Uandom, meaning a VlighW change in Whe codon VeTXence, VXch aV a change in Whe WhiUd
codon SoViWion, Zill VWill code foU Whe Vame, if noW VimilaU, amino acid. FXUWheUmoUe, Sol\SeSWide
chain V\nWheViV can be conWUolled b\ codonV WhaW do noW code foU VSecific amino acidV, bXW
inVWead VWoS and VWaUW WUanVlaWion. SWaUW codonV, VXch aV Whe AUG codon and, leVV fUeTXenWl\ Whe
GUG codon, Vignal Wo Whe UiboVome Wo begin mRNA WUanVlaWion Wo V\nWheViVe a Sol\SeSWide
chain. ComSaUaWiYel\, VWoS codonV, VXch aV UAG, UAA and UGA aUe XVed Wo WeUminaWe
Sol\SeSWide chain elongaWion.
When describing the genetic code, e[plain what is meant b\ the terms
µdegenerate¶ and µnon-random¶.
When deVcUibing Whe geneWic code, WeUmV VXch aV degeneUaWe and non-Uandom can be XVed Wo
deVcUibe amino acidV and Whe code iWVelf. The WeUm degeneUaWe iV XVed Wo deVcUibe Whe nXmbeU
of codon VeTXenceV WhaW can be XVed Wo code foU a VSecific amino acid. When an amino acid
haV moUe Whan one codon VeTXence, iW iV highl\ degeneUaWe. When an amino acid can onl\ be
coded b\ one codon iW iV noW degeneUaWe. FXUWheUmoUe, Whe WeUm non-Uandom iV XVed Wo deVcUibe
Whe aUUangemenW of Whe geneWic code Wable, aV lighW changeV in Whe codon VeTXence can aW WimeV
VWill VSecif\ Whe Vame oU VimilaU amino acid.
Describe the structure (secondar\ and tertiar\ levels) and function of tRNA
molecules.
WRNA molecXleV fXncWion Wo bUing Whe aSSUoSUiaWe amino acid Wo Whe UiboVome aW Whe aSSUoSUiaWe
codonV in Whe mRNA molecXle Wo Whe WRNA¶V comSlemenWaU\ anWicodonV. SecondaU\ WRNAV aUe
aUUanged in a µcloYeUleaf¶ VWUXcWXUe dXe Wo inWUamolecXlaU baVe SaiUing occXUUing ZiWhin Whe WRNA.
TeUWiaU\ WRNA VWUXcWXUeV aUe VimilaU Wo VecondaU\ VWUXcWXUeV, alWhoXgh Whe\ aUe µL-VhaSed¶ dXe Wo
VWacking inWeUacWionV and h\dUogen cUoVV-linkV. FXUWheUmoUe, WRNAV alVo conWain
SoVW-WUanVcUiSWional SUoceVVeV WhaW conWain VSecial modified baVeV. Some of WheVe
SoVW-WUanVcUiSWionall\ modified baVeV aUe belieYed Wo helS VWUengWhen Whe inWeUacWionV beWZeen
joining Whe amino acid Wo Whe WRNA, oU Wo VWUengWhen Whe inWeUacWion beWZeen Whe WRNA and Whe
codon on Whe mRNA aW Whe UiboVome.
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E[plain the process of tRNA aminoac\lation.
WRNA aminoac\laWion UeTXiUeV WZo UecogniWion VWeSV ZheUe Whe coUUecW amino acid mXVW fiUVW be
idenWified, and Whe coUUecW aminoac\l mXVW bind onWo an mRNA codon. DXUing Whe fiUVW
UecogniWion VWeS, aminoac\l-WRNA V\nWheWaVe (aaRS) mXVW VelecW Whe coUUecW amino acid Wo
aSSend Wo Whe 3¶ WeUminal UiboVe UeVidXe of iWV aVVociaWed WRNA, foUming an aminoac\l-WRNA
(aa-WRNA). aaRS iV able Wo UecogniVe VSecific WRNAV Wo chaUge ZiWh Whe coUUecW amino acid b\
UecogniVing Whe WRNA¶V XniTXe VWUXcWXUal feaWXUeV, VXch aV iWV acceSWoU VWem and anWicodon looS.
DXUing aminoac\laWion, Whe amino acidV mXVW fiUVW be acWiYaWed b\ ATP Wo foUm Whe acWiYaWion
SUodXcW, aminoac\l-aden\laWe. Aminoac\l-aden\laWe Zill When UeacW ZiWh Whe WRNA Wo foUm Whe
aa-WRNA. The aa-WRNA iV When able Wo bind onWo VSecific codonV on Whe mRNA Yia Whe aa-WRNA¶V
comSlemenWaU\ anWicodonV and Vignal Whe aSSUoSUiaWe amino acid Wo elongaWe Whe Sol\SeSWide
chain.
Describe the function of the ribosome during protein s\nthesis.
RiboVomeV aUe laUge comSle[eV of RNA and SUoWein WhaW aUe UeVSonVible foU acWing aV Whe ViWe of
SUoWein V\nWheViV and caWal\Ve SeSWide bond foUmaWion. The UiboVome fXncWionV b\ binding onWo
Whe mRNA Wo enVXUe Whe codonV SUeVenW on Whe mRNA can be Uead accXUaWel\ and moYe along
Whe mRNA Wo Whe VeTXenWial codon VeTXence. RiboVomeV conWain VSecific ViWeV foU WRNA Wo bind
Wo and acW aV a mediaWoU beWZeen Whe WRNA and mRNA Wo SUomoWe Sol\SeSWide chain iniWiaWion,
elongaWion and WeUminaWion.
Describe the ke\ structural features of the bacterial ribosome.
BacWeUial UiboVomeV aUe mainl\ comSoVed of UiboVomal RNA (URNA) and SUoWein and aUe
comSoVed of WZo VXbXniWV, a Vmall 30S URNA and laUge 50S VXbXniW of URNA Zhich conWainV
moUe URNA and SUoWeinV. The WRNA¶V anWicodonV Zill bind Wo Whe 30S VXbXniW of Whe UiboVome
ZhilVW Whe Uemaining SoUWionV of Whe WRNA Zill bind Wo Whe 50S VXbXniW. RiboVomal RNA haYe
comSle[ VecondaU\ VWUXcWXUeV WhaW cUeaWe a comSle[ WhUee-dimenVional UiboVome VWUXcWXUe WhaW
can foUm XniTXe VhaSeV. RiboVomeV alVo conWain WhUee WRNA binding ViWeV WhaW aVViVW in
WUanVlaWion, an aminoac\l (A) ViWe, SeSWid\l (P) ViWe, and an e[iW (E) ViWe. The A ViWe on a UiboVome
iV ZheUe Whe aminoac\l-WRNA Zill iniWiall\ aWWach Wo Whe UiboVome. The P ViWe iV ZheUe Whe
SeSWid\l-WRNA iV aWWached Wo Whe gUoZing Sol\SeSWide chain, and Whe E ViWe iV ZheUe Whe
deac\laWed-WRNA (no amino acid oU SeSWide) iV aWWached Wo eYenWXall\ e[iW fUom Whe UiboVome.
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WEEK 4
Lecture 9: Translation II
Describe the ke\ structural features of the eukar\otic ribosome.
The eXkaU\oWic UiboVome iV laUgeU and moUe comSle[ Whan bacWeUial UiboVomeV. Like Whe bacWeUial
UiboVome, Whe eXkaU\oWic UiboVome iV alVo comSoVed of a Vmall and laUge VXbXniW, alWhoXgh Whe\
aUe mXch laUgeU. EXkaU\oWic UiboVomeV conWain a Vmall 40S VXbXniW and a laUge 60S VXbXniW,
ZheUe Whe laUge VXbXniW Zill conWain moUe URNA and SUoWeinV Whan Whe Vmall VXbXniW. When
combined, Whe VXbXniWV make an 80S UiboVome.
Provide a brief overview of translation, including descriptions of the directions of
pol\peptide s\nthesis and ribosome movement.
DXUing WUanVlaWion, Sol\SeSWide V\nWheViV Zill SUoceed fUom Whe N-WeUminXV Wo Whe C-WeUminXV of
Whe amino acidV aV Whe UiboVome Zill Uead Whe mRNA in Whe 5¶ Wo 3¶ diUecWion. Chain elongaWion
occXUV dXUing WUanVlaWion, ZheUe a Sol\SeSWide chain Zill gUoZ b\ linking Whe chain ZiWh an
incoming WRNA¶V amino acid UeVidXe. FXUWheUmoUe, Sol\VomeV aUe UeTXiUed in boWh eXkaU\oWic
and bacWeUial acWiYe WUanVlaWion. Pol\VomeV enable mXlWiSle UiboVomeV Wo bind onWo a Vingle
mRNA WUanVcUiSW Wo SUodXce a µbead-on-a-VWUing¶ VWUXcWXUe called a Sol\UiboVome.
E[plain the process of peptide bond formation b\ the ribosomal peptid\l
transferase reaction.
PeSWide bond foUmaWion iV Whe SUoceVV ZheUe a SeSWide bond iV SUodXced Wo link WZo amino acidV
Wo one anoWheU and foUm a gUoZing SeSWide chain. PeSWide bond foUmaWion occXUV on Whe A, P
and E-ViWe of a UiboVome Yia Whe UiboVomal SeSWid\l WUanVfeUaVe UeacWion. DXUing WhiV UeacWion, a
SeSWid\l-WRNA iV aWWached Wo a NaVcenW Sol\SeSWide (gUoZing Sol\SeSWide chain) aW Whe UiboVomal
P-ViWe, ZhilVW an amino acid Zill bind Wo Whe WRNA aWWached Wo Whe A-ViWe on Whe UiboVome. A
nXcleoShilic aWWack-like UeacWion Zill When occXU on Whe fUee amino gUoXS, ZheUe Whe incoming
neZ amino acid Zill bUeak Whe caUbo[\lic bond WhaW aWWacheV Whe SeSWide chain Wo Whe WRNA on
Whe P-ViWe. The NaVcenW Sol\SeSWide Zill When diVSlace fUom Whe P-ViWe and WUanVfeU Wo Whe neZ
amino acid on Whe A-ViWe. ThiV diVSlacemenW leaYeV Whe WRNA in Whe P-ViWe ZiWh no amino acid oU
SeSWide chain, ZhilVW Whe A-ViWe noZ haV Whe gUoZing Sol\SeSWide chain boXnd Wo Whe neZ amino
acid WhaW had been inWUodXced. The UiboVome Zill When moYe onWo Whe ne[W codon along Whe
mRNA, caXVing Whe ViWeV Wo moYe oYeU b\ one and Whe c\cle Wo UeSeaW.
Describe the structure and function of the Shine-Dalgarno sequence in bacteria.
The Shine-DalgaUno VeTXence in bacWeUia iV a VeTXence WhaW iV comSlemenWaU\ beWZeen a Vmall
SaUW of mRNA and SaUW of Whe UiboVome ZheUe Whe 16S UURNA of Whe UiboVome. The
Shine-DalgaUno VeTXence in Whe mRNA conWainV a loW of SXUineV, ZhilVW Whe Shine-DalgaUno
VeTXence in Whe UiboVome conWainV a loW of comSlemenWaU\ S\UimidineV. DXe Wo Whe VWaUW codon,
AUG, alVo being able Wo code foU Whe amino acid MeW, Whe UiboVome mXVW be able Wo UecogniVe
Zhen SUoWein WUanVlaWion VhoXld begin oU Zhen MeW mXVW be Vignalled. The SXUSoVe of Whe
Shine-DalgaUno VeTXence iV Wo cUeaWe a diffeUenWiaWion foU Whe AUG codon, aV iW SeUmiWV Whe
UiboVome Wo bind Wo Whe coUUecW iniWiaWion codon and begin WUanVlaWion.
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Describe the main features of each of the three main stages of translation: chain
initiation, elongation and termination.
The WhUee main VWageV of WUanVlaWion inclXde chain iniWiaWion, elongaWion and WeUminaWion.
The fiUVW VWage of WUanVlaWion iV chain iniWiaWion, ZheUe a MeW-WRNA iV N-foUm\laWed Wo foUm
fMeW-WRNAfMeW and begin Whe SUoceVV of WUanVlaWion. TUanVlaWion iniWiaWion in bacWeUia UeTXiUeV boWh
UiboVomal VXbXniWV Wo aVVemble ZiWh Whe fMeW-WRNAfMeW on a coUUecWl\ aligned mRNA, foUming a
comSle[ WhaW can commence chain elongaWion. fMeW-WRNAfMeW Zill When occXS\ Whe P-ViWe of Whe
UiboVome, ZhilVW Whe A-ViWe iV XnoccXSied and able Wo acceSW incoming aminoac\l-WRNA.
IniWiaWion facWoUV WhaW aUe noW aVVociaWed ZiWh Whe UiboVome, VXch aV iniWiaWion-facWoU I, II and III
aUe alVo UeTXiUed dXUing chain iniWiaWion.
The Vecond VWage of WUanVlaWion iV chain elongaWion, ZheUe Whe UiboVome addV amino
acidV Wo a gUoZing Sol\SeSWide chain in a WhUee UeacWion c\cle. Decoding iV Whe fiUVW UeacWion in
Whe elongaWion c\cle, ZheUe Whe UiboVome Zill XVe GTP Wo VelecW and bind Wo an aminoac\l-WRNA
WhaW conWainV anWicodonV WhaW aUe comSlemenWaU\ Wo Whe mRNA¶V codon in Whe UiboVome¶V A-ViWe.
TUanVSeSWidaWion iV Whe Vecond VWage ZheUe SeSWide bond foUmaWion Zill occXU. DXUing WhiV
UeacWion, Whe WRNA in Whe P-ViWe¶V SeSWid\l gUoXS iV WUanVfeUUed Wo Whe aminoac\l gUoXS in Whe
A-ViWe. The final UeacWion in elongaWion iV WUanVlocaWion, ZheUe Whe UiboVome Zill XVe GTP Wo
moYe onWo Whe ne[W codon VeTXence in Whe mRNA. DXUing WhiV UeacWion, Whe ViWeV in Whe
UiboVome Zill VhifW oYeU b\ one, meaning Whe A-ViWe Zill become Whe P-ViWe, and Whe P-ViWe Zill
become Whe E-ViWe. Once Whe UiboVome moYeV and Whe P-ViWe becomeV Whe E-ViWe, Whe
deac\laWed-WRNA Zill When e[iW Whe UiboVome and Whe elongaWion c\cle Zill UeSeaW.
The final VWage of WUanVlaWion iV chain WeUminaWion, ZheUe elongaWion of Whe Sol\SeSWide
chain iV ceaVed once Whe UiboVome haV Ueached Whe mRNA¶V VWoS codon. Once Whe UiboVome
haV Ueached a VSecific VWoS codon, a VSecific UeleaVe facWoU (RF) Zill bind Wo Whe codon aW Whe
A-ViWe of Whe UiboVome. EiWheU RF-1 oU RF-2 Zill be iniWiall\ UeleaVed deSending on Whe VWoS
codon. The UeleaVe facWoU Zill When caXVe Whe Sol\SeSWide chain Wo h\dUol\Ve and be UeleaVed
fUom Whe SeSWid\l WRNA in Whe P-ViWe. AnoWheU UeleaVe facWoU, RF-3 Zill When bind ZiWh Whe SUodXcW
of Whe h\dUol\ViV, GDP, Wo Whe P-ViWe and e[change iWV GDP foU GTP, caXVing Whe iniWial RF Wo be
UeleaVed. RF-3 Zill h\dUol\Ve iWV boXnd GTP, UeleaVing boWh Whe SUodXcW and RF-3, ZhilVW a
UiboVome Uec\cling facWoU (RRF) bindV Wo Whe A-ViWe. The WZo VXbXniWV of Whe UiboVome Zill When
VeSaUaWe fUom each oWheU and WhXV fUom Whe mRNA, Uead\ Wo be UeaVVembled Wo UeSeaW Whe
SUoceVV.
E[plain wh\ and how post-translational processing events occur.
PoVW-WUanVlaWional SUoceVVing occXUV Wo enVXUe Whe Sol\SeSWide chain can be made inWo a
fXncWional SUoWein. Pol\SeSWide chainV aUe noW in WheiU final folded foUm afWeU WUanVlaWion noU aUe
Whe\ locaWed in WheiU WaUgeWed cellXlaU deVWinaWion, meaning Whe\ aUe noW in WheiU final fXncWional
foUm. PoVW-WUanVlaWional SUoceVVing occXUV ZiWh Whe aid of oWheU SUoWeinV, VXch aV
UiboVome-aVVociaWed chaSeUoneV, Wo aVViVW in SUoWein folding once Whe Sol\SeSWide chain iV
UeleaVed fUom Whe UiboVome. FXUWheUmoUe, naVcenW Sol\SeSWideV can achieYe WheiU maWXUe foUm
b\ XndeUgoing SoVW-WUanVlaWional SUoceVVeV VXch aV SUoWeol\ViV, coYalenW modificaWion,
WUanVlocaWion, and gl\coV\laWion. DXUing SUoWeol\ViV, Whe laUgeU Sol\SeSWide iV cleaYed inWo
VmalleU Sol\SeSWide chainV. DXUing coYalenW modificaWion, chemical gUoXSV can be aWWached Wo a
SaUWicXlaU Sol\SeSWide chain, ZhilVW in gl\coV\laWion, caUboh\dUaWe gUoXSV aUe added Wo SUoWeinV
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foU YaUioXV fXncWionV. TUanVlocaWion enableV Whe SUoWein Wo moYe WhUoXgh Whe membUane,
enabling iW Wo Ueach iWV cellXlaU deVWinaWion and indXce SUoWein fXncWion.
List two antibiotics that block the process of translation and briefl\ describe their
mechanism of action.
TZo anWibioWicV WhaW can block Whe SUoceVV of WUanVlaWion inclXde VWUeSWom\cin and
chloUamShenicol. SWUeSWom\cin iV an aminogl\coVide WhaW, Zhen adminiVWeUed in loZ doVeV,
fXncWionV b\ caXVing Whe UiboVome Wo incoUUecWl\ Uead Whe mRNA. ThiV inhibiWV Whe gUoZWh of Whe
VXVceSWible cell, bXW onl\ killV Whe cell Zhen adminiVWeUed aW higheU concenWUaWionV, ZheUe
VWUeSWom\cin Zill SUeYenW SUoSeU chain iniWiaWion. A lack of fXncWional SUoWeinV inhibiWV Whe bacWeUia
fUom fXncWioning, enabling Whe immXne V\VWem Wo effecWiYel\ deVWUo\ iW. FXUWheUmoUe,
chloUamShenicol inWeUfeUeV ZiWh Whe en]\maWic acWiYiW\ of SeSWid\l WUanVfeUaVe b\ binding Wo Whe
laUge VXbXniW neaU Whe A-ViWe, WheUeb\ SUeYenWing Whe foUmaWion of SeSWide bondV.
ChloUamShenicol¶V clinical XVeV, hoZeYeU, aUe limiWed aV iW can caXVe Wo[ic Vide effecWV Wo Whe
hoVW.
Lecture 10: Review of Protein Structure
Describe the properties and chemical characteristics of the side chains that
distinguish the standard 20 amino acids.
All Į-amino acidV conWain a geneUal foUmXla conViVWing of one amino gUoXS, one caUbo[\l gUoXS,
and one Vide chain gUoXS. The Vide chain gUoXS, alVo knoZn aV R-gUoXSV, in Whe amino acid iV
ZhaW deWeUmineV Whe W\Se of amino acid. Side chainV haYe YaU\ing SUoSeUWieV WhaW aUe deSendenW
on WheiU fXncWion. The\ can be non-SolaU, SolaU, acidic oU baVic. Non-SolaU Vide chainV aUe
h\dUoShobic, meaning Whe\ deWeU fUom ZaWeU, aV Whe\ conWain loWV of h\dUocaUbonV.
ComSaUaWiYel\, SolaU Vide chainV aUe h\dUoShilic, meaning Whe\ aUe ZaWeU loYing, aV Whe\ conWain
loWV of h\dUocaUbon, h\dUo[\l gUoXSV and VXlfh\dU\l gUoXSV. TheVe gUoXSV haYe a gUeaWeU
diffeUence in elecWUonegaWiYiW\ in WheiU bondV WhaW conWUibXWe Wo Whe SolaU naWXUe of Whe Vide chainV.
FXUWheUmoUe, acidic Vide chainV aUe h\dUoShilic and negaWiYel\ chaUged aV Whe\ conWain caUbo[\l
gUoXSV, ZhilVW baVic Vide chainV aUe SoViWiYel\ chaUged aV Whe\ conWain amino gUoXSV.
E[plain the process of peptide bond (and pol\peptide chain) formation.
PeSWide bond foUmaWion occXUV Yia a deh\dUaWion UeacWion beWZeen Whe caUbo[\l gUoXS of one
amino acid, and Whe amino gUoXS of a VeTXenWial amino acid. DXUing Whe deh\dUaWion UeacWion,
Zhen Whe amino-gUoXS of a fUee amino acid joinV onWo a caUbo[\l gUoXS of an amino acid in a
gUoZing Sol\SeSWide chain, a H20 molecXle iV UeleaVed, foUming a SeSWide bond. GUoZWh of a
SeSWide chain iV in Whe amino N-WeUminXV Wo caUbo[\l C-WeUminXV diUecWion, aV VXbVeTXenW
SeSWide bondV aUe added Wo Whe C-WeUminXV. Amino acid Vide chainV alVo SUojecW fUom Whe
UeSeWiWiYe backbone of Whe final Sol\SeSWide.
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Describe the four levels of protein structure (including the various bonds and
forces involved at each level) and e[plain the differences between fibrous and
globular proteins.
The foXU leYelV of SUoWein VWUXcWXUe inclXde SUimaU\, VecondaU\, WeUWiaU\ and TXaWeUnaU\ VWUXcWXUe.
PUimaU\ VWUXcWXUe iV Whe VimSle foUm of SUoWein VWUXcWXUe, ZheUe a XniTXe VeTXence of
amino acidV iV linked b\ SeSWide bondV Wo foUm a lineaU Sol\SeSWide chain. The VeTXence of
amino acidV in SUimaU\ SUoWein VWUXcWXUe iV deWeUmined b\ geneWic infoUmaWion.
The VecondaU\ VWUXcWXUe of a SUoWein occXUV Zhen SaUWV of Whe Sol\SeSWide chain can fold
inWo iUUegXlaU VhaSe, indXcing a confoUmaWional change VXch aV WXUnV oU looSV. DeSending on
Whe h\dUogen bonding beWZeen Whe caUbo[\l and amino gUoXS of Whe Sol\SeSWide chain, WZo
majoU elemenWV of VecondaU\ SUoWein VWUXcWXUe inclXde Whe Į-heli[ and Whe ȕ-VheeW, ma\ foUm.
The Į-heli[ VWUXcWXUe onl\ inYolYeV one Sol\SeSWide chain WhaW UighW-handedl\ WXUnV eYeU\ 3.6
amino acidV VWabiliVed b\ inWeUnal h\dUogen bondV. The Į-heli[ VWUXcWXUe beginV fUom Whe
N-WeUminal end of Whe Sol\SeSWide chain, ZheUe Whe h\dUogen bondV occXU beWZeen Whe caUbo[\l
bondV of one amino acid, and Whe N±H bond of an amino acid foXU SoViWionV aZa\. DXUing WhiV
VWUXcWXUe, Whe R-gUoXSV of Whe amino acidV all SUojecW oXWZaUdV fUom Whe heli[. FXUWheUmoUe,
ȕ-VheeW VWUXcWXUe comSaUaWiYel\ inYolYeV one oU moUe Sol\SeSWide chainV WhaW can looS in a
SaUallel oU anWiSaUallel diUecWion. In SaUallel ȕ-VheeWV, Sol\SeSWide UegionV aUe lined XS in Whe
Vame SolaUiW\/diUecWion, meaning iW UeTXiUeV looSing Wo bUing a Vingle Sol\SeSWide WogeWheU. The
h\dUogen bonding in SaUallel ȕ-VheeWV iV TXiWe angled, SUoYiding leVV VWabiliW\. In anWiSaUallel
ȕ-VheeWV, Sol\SeSWide UegionV aUe lined XS in diffeUenW SolaUiWieV/diUecWionV, meaning Whe
Sol\SeSWide Uegion VimSl\ UeTXiUeV Whe Sol\SeSWide Wo WXUn Wo bUing a Vingle Sol\SeSWide WogeWheU.
H\dUogen bonding in anWiSaUallel ȕ-VheeWV iV moUe lineaU and WheUefoUe moUe VWable Whan SaUallel
ȕ-VheeWV.
The WeUWiaU\ VWUXcWXUe iV Whe final fXll\ folded foUm of a SUoWein, ZheUe iUUegXlaU conWoUWionV
dXe Wo inWeUacWionV beWZeen diffeUenW Vide chainV cUeaWe XniTXe confoUmaWional changeV. In
WeUWiaU\ SUoWein VWUXcWXUeV, Whe h\dUoShobic Vide chainV aUe XVXall\ locaWed ZiWhin Whe SUoWein,
ZhilVW Whe h\dUoShilic Vide chainV aUe foXnd on Whe e[WeUioU of Whe SUoWein. DeVSiWe WeUWiaU\
VWUXcWXUeV conWaining VeYeUal Zeak inWeUacWionV, VXch aV h\dUoShobic inWeUacWionV, H-bondV and
ionic bondV, Whe cXmXlaWiYe effecW of Whe inWeUacWionV giYeV Whe SUoWein iWV VSecific VhaSe and
incUeaVed VWabiliW\. SWUongeU inWeUacWion, VXch aV diVXlShide bUidgeV, incUeaVe VWabiliW\ and
SUodXce VWUong coYalenW bondV beWZeen Vide chainV and WZo c\VWeine amino acidV.
QXaWeUnaU\ VWUXcWXUeV aUe Whe final fXll\ folded foUm of a SUoWein WhaW conViVWV of moUe
Whan one Sol\SeSWide chain held WogeWheU b\ non-coYalenW bondV, called a VXbXniW. QXaWeUnaU\
SUoWeinV can be chaUacWeUiVed aV being fibUoXV oU globXlaU. FibUinoXV TXaWeUnaU\ SUoWeinV aUe
long, e[Wended and Uod-like VhaSed SUoWeinV WhaW aUe W\Sicall\ inVolXble in ZaWeU dXe Wo WheiU high
Uigid VWUXcWXUeV WhaW moVWl\ conWain Į-heliceV. FibUinoXV SUoWeinV inclXde collagen, keUaWin and
fibUin. ComSaUaWiYel\, globXlaU TXaWeUnaU\ SUoWein VWUXcWXUeV aUe comSle[, comSacW and VSheUical
SUoWeinV WhaW fold back in on WhemVelYeV. GlobXlaU SUoWeinV conWain a h\dUoShilic VXUface and
h\dUoShobic coUe and aUe W\Sicall\ VolXble in ZaWeU aV Whe\ conWain boWh Į-heliceV and ȕ-VheeWV.
An e[amSle of a globXlaU SUoWein inclXdeV haemoglobin.
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Describe the combined forces/effects that contribute to protein folding and
stabilit\.
The combined foUceV/effecWV WhaW conWUibXWe Wo SUoWein folding inclXde Whe h\dUoShobic and
elecWUoVWaWic effecWV. The h\dUoShobic effecW iV Whe majoU deWeUminanW of SUoWein VWUXcWXUe ZheUe
h\dUoShobic amino acidV Zill inWeUacW ZiWh each oWheU, bXW noW ZiWh ZaWeU oU oWheU amino acidV.
DXe Wo ZaWeU commonl\ being SUeVenW in Whe enYiUonmenW, h\dUoShobic amino acidV aUe foXnd in
Whe inWeUioU of globXlaU SUoWeinV, ZhilVW Whe h\dUoShilic amino acidV aUe foXnd on Whe VXUface of
Whe SUoWein. The likelihood of Whe locaWion of an amino acid can be SUedicWed b\ iWV h\dUoSaWh\,
ZheUe Whe higheU Whe h\dUoSaWh\, Whe moUe likel\ Whe amino acid Zill be locaWed in Whe inWeUioU of
Whe SUoWein. ComSaUaWiYel\, Whe loZeU Whe h\dUoSaWh\, Whe moUe likel\ Whe amino acid Zill be
locaWed on Whe e[WeUioU of Whe SUoWein. ElecWUoVWaWic effecWV aUe anoWheU deWeUminanW of SUoWein
VWUXcWXUe, alWhoXgh Wo a leVVeU e[WenW aV iW acWV oYeU Vmall diVWanceV Wo µfine WXne¶ VWUXcWXUeV
dXUing SUoWein folding. ElecWUoVWaWic effecWV inclXde h\dUogen bondV in Whe backbone and Vide
chainV, ion SaiUV/ValW bUidgeV beWZeen chaUged Vide chainV, oU inWeUacWionV beWZeen meWal ionV
VXch aV ]inc.
Describe the features of various structural motifs commonl\ found in
DNA-binding proteins such as transcription factors.
DNA binding SUoWeinV aUe neceVVaU\ foU UegXlaWing DNA UeSlicaWion, UeSaiU and WUanVcUiSWion b\
inWeUacWing diUecWl\ ZiWh Whe DNA doXble heli[. MoVW DNA binding SUoWeinV can be claVVed b\
VWUXcWXUal moWifV Whe\ XVe Wo aWWach Wo Whe majoU gUooYe of a DNA doXble heli[. TUanVcUiSWion
facWoUV aUe a foUm of DNA binding SUoWein WhaW alVo inWeUacWV ZiWh oWheU SUoWein facWoUV Wo UegXlaWe
SUoWein V\nWheViV. SWUXcWXUal moWifV fUeTXenWl\ conWained ZiWhin WUanVcUiSWion facWoUV inclXde
heli[-WXUn-heli[, ]inc fingeU and leXcine ]iSSeU moWifV.
Heli[-WXUn-heli[ moWifV aUe comSoVed of aSSUo[imaWel\ 20 amino acidV in Whe foUm of WZo
VhoUW Į-helical VegmenWV. Each Į-heli[ iV aUoXnd 8 amino acidV long and aUe VeSaUaWed b\ a ȕ
WXUn. One VegmenW of Whe heli[ iV Whe UecogniWion heli[ Zhich SUoWUXdeV fUom Whe SUoWein
VWUXcWXUe, conWaining man\ of Whe amino acidV WhaW Zill inWeUacW ZiWh Whe majoU gUooYe of a DNA
doXble heli[.
LeXcine ]iSSeU moWifV conWain WZo Į-heliceV WhaW foUm a coiled coil WhaW iV mainWained b\
leXcine UeVidXe WhaW µ]iSSeUV¶ Whe WoS SaUW of Whe moWif Wo enVXUe iW VWa\V WogeWheU. The boWWom SaUW
of a leXcine ]iSSeU doeV noW haYe an\ leXcine UeVidXe aV Whe WZo coilV of Whe ]iSSeU Zill diUecWl\
bind Wo Whe majoU gUooYe of Whe DNA.
Zinc fingeU moWifV conWain WhUee ]inc fingeUV fUom a UegXlaWoU\ SUoWein comSle[ed ZiWh
DNA. Each ]inc fingeU cooUdinaWeV ZiWh WZo hiVWidine and WZo c\VWeine amino acid UeVidXeV.
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