replication and protein synthesis Flashcards

1
Q

state some features and constraints of DNA polymerase

A

Template directed
High fecundity- very low error rate
~1 mistake per 10^9 nucleotides
Must be able to replicate massive amounts of DNA in a few hours.

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2
Q

DNA polymerase I

Discovery (A. korberg, 1958) relied on:

A

observation that deoxyribonucleoside 5’ triphosphates (dATP, dCTP, dGTP, dTTP) are heat-stable precursors of DNA synthesis.

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3
Q

How is DNA polymerase 1 isolated

A

Break ecoli cells
centrifuge into layers
test layers with dTTP cooperation assay

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4
Q

DNA polymerase Requirements for synthesis:

A

Single stranded template
dNTP building blocks
Primer can be RNA or DNA

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5
Q

which Direction do DNA polymerases work on a Template strand

A

5’–>3’

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6
Q

Describe the consequences of unidirectionality of DNApol on DNA Synthesis

A

As a consequence of this one strand of DNA is called the lagging strand asn is done in small fragments called ozeki fragments.
Newly formed DNA will be found in ~1000 fragments which are incorporated with time.

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7
Q

how are RNA primers removed?

A

Rnase H removes RNA primers

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8
Q

what is the function of DNA ligase

A

DNA ligase joins ozeki fragments

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9
Q

what are the functions of DNA polymerases

A
  • > chromosome replication
  • > DNA repair
  • > DNA recombination
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10
Q

when is the new DNA strand checked

A

Before adding another nucleotide previous additions are checked if one is mismatched phosphodiester bond is cut and nucleotide replaced.

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11
Q

how does proof reading affect DNA polymerase acuracy? how is it done?

A

Replication DNA polymerases have built in proofreading ability; thus can remove mis-incorporated nucleotides and try again.
For eukaryotic DNA replication: error rate without proofreading: 1 in ~10^5
with proofreading: 1 in ~10^7
with proofreading and mismatch repair: 1 in ~10^9
Proofreading active site is the endonuclease active site
Before adding another nucleotide previous additions are checked if one is mismatched phosphodiester bond is cut and nucleotide replaced.

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12
Q

compare DNA replication in eukaryotes vs bacteria

A

replication can be continuous in bacteria where as happens in ‘s’ phase in eukaryotes

single origin in bacteria multiple in euk

circular DNA no telomeres, euk telomeres special end replication mech

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13
Q

what is the problem with replicating the lagging strand end of a chromosome and how is this problem resolved?

A

No 3’ hydroxyl group to replicate laging end
Without tylermorase chromosomes would get progressively shorter.
Telomeres repete themselves 500-3000 times at end of chromosome

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14
Q

why is having multiple origins beneficial for a genome?

A

allow replication of large genomes in a short time

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15
Q

what do telomerases do?

A

Telomerases add additional bases to the end of the chromosome
DNA polymerase Alpha completes the new strand thus completely copying the chromosome

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16
Q

what does miniture RNA (miRNA) do

A

miniature RNA regulates RNA

17
Q

Why is thymine used not uracil in DNA as well as RNA

A

Use of t important because at pH7 C will occasionally spontaneously deaminate into a U so our body would have a hard time recognizing damaged deaminated DNA if T was not used.

18
Q

What direction do RNA polymerases work in?

A

RNA polymerases like DNA polymerases work in the 5’ to 3’ direction

19
Q

RNA polymerase requires

A

NTPs- nucleotide triphosphate.

Double stranded helix but no primer.

20
Q

why is their another highly conserved region of DNA upstream of the promoter region in bacteria

A

found to be a result of the structure of RNA pol as there are two spaced out binding sites

21
Q

how is excessive tightening of the DNA double helix prevented before strands are seperated

A

To prevent DNA helicase tightening adjacent areas of the double helix too much to unwind accessory proteins must go along and loosen the super helical structure of the DNA before transcription.

22
Q

Eucaryotic poll 2 cannot initiate transcription without presence of general transcription factors:

A

TFIID- (14 subunit structure) dictates start of transcription

TFIIH-(10 subunits) includes helicase DNA unwinding and includes helicase activity.

23
Q

How did Early insight from mutagen studies in tobacco mosaic virus demonstrate that the code was non overlapping. Used as early evidence for triplet code

A

Addition of a base was shown to scramble code
Removal of base was demonstrated to return phenotype to wildtype
3/+ mutations shown to have no (little) effect on the organism’s phenotype
(Crick, F., Brenner, S., et al. (1961). Nature 192, 1227-1232)

24
Q

why does RNA extend beyond 5’ and 3’ stop and start codons?

A

enables regulatory functions

25
Q

Bacterial requirements for protein synthesis:

A
mRNA
tRNA deliver aminoacid
GTP
Additional bacterial proteins (mostly GTPases)
Elongation factors 
Termination factors
26
Q

how are stop codons recognized in ribosomes

A

No tRNA can read stop codons instead they are recognised by protein ‘release factors’

27
Q

comment on the difference between eukaryotic and prokaryotic ribosomes

A

pro- 60s
euk- 80s both subunits larger

the ‘s’ is about sedimentation speed the higher the quicker the speed