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Lecture 4

Lecture 4 CSB351 Virology

3 Pages
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Department
Cell and Systems Biology
Course Code
CSB351Y1
Professor
Mounir Abou Haidar

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Nucleic Acid Enzymes
Polymerase: enzyme that synthesize N.A.
{
Pyrophosphate: need triphosphate (cytosine triphosphate CTP, adenine triphosphate ATP, etc )-->triphosphate, broken down by
polymerase, energy from pyrophosphate used to make covalent bond, RNA/DNA synthesized becomes a chain-like molecule
(covalent bonds, very strong)
DNA-dependent RNA polymerase: (DNA viruses)
a.
Transcription of mRNA from nuclear DNA
Need promoter: sequence of DNA that RNA polymerase binds to--> to start transcription
Weak/strong promoter: how much it attracts RNA polymerase
Virus makes their promoters more attractive than that of the cell, so can use cell's machinery to transcribe their own
genes --> thus strong promoters are usually viral
RNA-dependent RNA polymerase:(RNA viruses)
b.
Make new RNA from RNA template (copyase)
RNA Viruses code for their own, for their own replication
ALL RNA viruses encode for this
RNA polymerase: Doesn't need primer
{
DNA-dependent DNA polymerase: needs primer
{
Most DNA viruses code for the own, make new DNA copy from RNA template (reverse transcriptase)
Retroviruses!
P 13:
Modifying Enzymes
Methylases
Modifying enzymes that add a methyl (-CH3) group --> modify the first nucleotide on the RNA (mRNA or (+) viral RNA)--> first
nucleotide normally is guanosine, at position 7 (m7G)--> add methyl group
{
Viral (+) RNA: majority of these +RNA viruses have a cap at 5' end as well, like the mRNAs (some don't--come back later:
polio)
¾
*MOST (+) RNA viruses have 5' cap on their mRNA!!
*Polio: (+) virus, has no cap! Why? Diff strategy: creates special structure--> cap-independent translation! (majority are cap-
dependent)
Viral (-) RNAviruses: not translatable, don't need cap, thus not capped
¾
DNA viruses: can't put cap on DNA --> DNA not translatable by ribosome (only translates RNA)
¾
Need RNA polymerase of the cell to read DNA and transcribe in the nucleus into mRNA (which usually transcribe DNA of the
cell)--> this is why DNA needs to go into nucleus for viral replication, because need cell's mechanisms and polymerases
Get special structure at 5' end of all mRNA of cell--> makes ribo recognize them, is a "cap", like signal --> ready for translation!
{
Ligases:
Repairs "broken" nucleotides
{
Repairs circular DNA from a linear chain
{
Don't worry about terminal tranferases
{
Nucleases:
Endonucleases: Restriction enzymes -->enzymes can cut in the middle of the chain
1.
Cuts only double-stranded DNA
Sequences are always symmetrical: 2-fold symmetry
Staggered breaks--> complementary cohesive ends
Exonucleases: cuts from one end of the nucleic acid chain or the other (i.e. 5' end)
2.
Rnase (ribonucleases): digests Rna (*DICER)
Dnase (deoxyribonucleases): digests DNA
E.g. Snake venome phosphdiesterase splits RNA from 3' end sequentially, have same reaction for DNA
Nuclease S1: cuts only single-stranded regions of DNA or RNA (S1 = single-stranded)
3.
RnaseH: Ribonuclease H = hybrid
4.
Only found in RETROVIRUSES (i.e. HIV)
Retroviruses have RNA as genome --> virus first synthesize cDNA from RNA --> now have RNA/DNA hybrid
Rnase H cuts RNA ONLY when it's hybridized with DNA --> after RNA copied into DNA, is degraded [while DNA is copied into
DS DNA (DNA polymerase: DNA-dependent DNA polymerase]
Degrades N.A.
{
Helicases:
Responsible for unwinding enzymes or proteins
{
Large majority of viral polymerases (RNA/DNA): have helicase activity
{
Lecture 4: Pg 12 cont'd
September-24-09
2:10 PM
CSB351 Course Notes Page 1
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Description
Lecture4: Pg12contd September-24-09 2:10 PM NucleicAcidEnzymes { Polymerase:enzymethatsynthesizeN.A. Pyrophosphate:needtriphosphate(cytosinetriphosphateCTP,adeninetriphosphateATP,etc)-->triphosphate,brokendownby polymerase,energyfrompyrophosphateusedtomakecovalentbond,RNADNAsynthesizedbecomesachain-likemolecule (covalentbonds,verystrong) { RNApolymerase:Doesntneedprimer a. DNA-dependentRNApolymerase: (DNAviruses) TranscriptionofmRNAfromnuclearDNA Needpromoter:sequenceofDNAthatRNApolymerasebindsto-->tostarttranscription Weakstrongpromoter:howmuchitattractsRNA polymerase Stronger=transcribingmoreRNA Virusmakestheirpromotersmoreattractivethanthatofthecell,socan usecellsmachinerytotranscribetheirown genes-->thusstrongpromotersareusuallyviral b. RNA-dependentRNApolymerase:(RNAviruses) MakenewRNAfromRNAtemplate(copyase) RNAVirusescodefortheirown,fortheirownreplication ALLRNAvirusesencodeforthis { DNA-dependentDNApolymerase:needsprimer MostDNAvirusescodefortheown,makenewDNA copyfrom RNAtemplate(reversetranscriptase) Retroviruses! P13: ModifyingEnzymes Methylases { Modifyingenzymesthataddamethyl(-CH3)group-->modifythefirstnucleotideontheRNA(mRNAor(+)viralRNA)-->first nucleotidenormallyisguanosine,atposition7(m7G)-->addmethylgroup { Getspecialstructureat5 endofall mRNAofcell-->makesriborecognizethem,isacap,likesignal -->readyfortranslation! Viral(+)RNA: majorityofthese+RNAviruseshaveacap at 5 endas well,likethemRNAs (somedont--comebacklater: polio) *MOST(+)RNAviruseshave5cap on theirmRNA!! *Polio:(+)virus,hasnocap! Why?Diffstr
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