replication and protein synthesis Flashcards
state some features and constraints of DNA polymerase
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.
DNA polymerase I
Discovery (A. korberg, 1958) relied on:
observation that deoxyribonucleoside 5’ triphosphates (dATP, dCTP, dGTP, dTTP) are heat-stable precursors of DNA synthesis.
How is DNA polymerase 1 isolated
Break ecoli cells
centrifuge into layers
test layers with dTTP cooperation assay
DNA polymerase Requirements for synthesis:
Single stranded template
dNTP building blocks
Primer can be RNA or DNA
which Direction do DNA polymerases work on a Template strand
5’–>3’
Describe the consequences of unidirectionality of DNApol on DNA Synthesis
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.
how are RNA primers removed?
Rnase H removes RNA primers
what is the function of DNA ligase
DNA ligase joins ozeki fragments
what are the functions of DNA polymerases
- > chromosome replication
- > DNA repair
- > DNA recombination
when is the new DNA strand checked
Before adding another nucleotide previous additions are checked if one is mismatched phosphodiester bond is cut and nucleotide replaced.
how does proof reading affect DNA polymerase acuracy? how is it done?
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.
compare DNA replication in eukaryotes vs bacteria
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
what is the problem with replicating the lagging strand end of a chromosome and how is this problem resolved?
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
why is having multiple origins beneficial for a genome?
allow replication of large genomes in a short time
what do telomerases do?
Telomerases add additional bases to the end of the chromosome
DNA polymerase Alpha completes the new strand thus completely copying the chromosome
what does miniture RNA (miRNA) do
miniature RNA regulates RNA
Why is thymine used not uracil in DNA as well as RNA
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.
What direction do RNA polymerases work in?
RNA polymerases like DNA polymerases work in the 5’ to 3’ direction
RNA polymerase requires
NTPs- nucleotide triphosphate.
Double stranded helix but no primer.
why is their another highly conserved region of DNA upstream of the promoter region in bacteria
found to be a result of the structure of RNA pol as there are two spaced out binding sites
how is excessive tightening of the DNA double helix prevented before strands are seperated
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.
Eucaryotic poll 2 cannot initiate transcription without presence of general transcription factors:
TFIID- (14 subunit structure) dictates start of transcription
TFIIH-(10 subunits) includes helicase DNA unwinding and includes helicase activity.
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
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)
why does RNA extend beyond 5’ and 3’ stop and start codons?
enables regulatory functions
