DNA Replication Flashcards
How does DNA synthesis by DNA polymerase work?
DNA polymerases cannot initiate DNA replication with just ‘ a template and dNTPd
They extend existing chains - from a primer - template substrate
- all works in the 5’ to 3’ direction
what is polymerase III (Pol III)?
Replicative polymerase
What is the speed and error of DNA replication in E. coli?
Template directed DNA synthesis is up to 1000 nucleotides per second
- has a very small error rate of 10^-9 - 10^-10
-the initial Pol III error rate is higher but it is proof-read by intrinsic exonuclease
- post-replication mismatch repair also reduced error
What allows both DNA strands to be replicated at once?
Semi-discontinuous DNA replication allows both strands to be replicated at a single replication forrk
- both in the 5’ - 3’ direction, leading strand can be replicated continuously
- lagging strand is not, creates okazaki fragments
What does DNA polymerase III require for DNA synthesis?
a primer
What are the roles of DNA primase and DNA ligase
DNA primase - Makes successive short (3-5 nucleotide) RNA primers on the lagging strand template (Okazaki fragments)
RNA is later removed by DNA Polymerase I and replaced with DNA
DNA ligase - Then joins together the Okazaki fragments
How is DNA unwound?
The combined action of DnaB and SSB proteins.
What is SSB?
Single-stranded DNA binding protein
What is DnaB
DNA helicase
What does the beta sliding clamp do?
Keeps Pol III on the DNA (t1/2 = 100 minutes)
What is a Linking Number (Lk)?
The number of times one strand winds around the other.
What is the Linking Number equation for relaxed DNA?
Lk^o = N/h
N = the number of base pairs
h = the helical repeat in solution = 10.5 bp/turn
σ = ΔLk/Lk^o
What is over/under winding and how does it effect DNA replication/RNA transcription?
Overwound DNA - positively superoiled
Underwound DNA - negatively supercoiled
- all positive and negative supercoils must be removed or replication/transcription will stop. To do this requires continuous DNA breakage and strand passage events
How does the linking number (Lk) of parental DNA strands change after replication?
Reduces to zero
How can Parental DNA strands be resolved after replication and what enzyme catalyses the process?
By DNA breakage and strand passage events which change the linking number (Lk) - catalysed by DNA topioisomerase
How does DNA topoisomerase work to resolve topological complexity?
The linking number of closed circular double stranded DNA (dsDNA) cannot be changed without one or both strands being broken.
DNA topoisomerease change the linking number of DNA in vivo by making single or double stranded breaks
- they then pass a segment off ssDNA or dsDNA through the break
-they then rejoin the DNA ends
What direction is Circular DNA replicated in?
bidirectionally
(also 5’ - 3’ obviously)
How is the oriC (origin) of E. coli replication determined?
oriC (origin) recognised by DnaA
DnaA binds to DnaA boxes with a consensus sequence, further DnaA molecules bind to form a filament leading to unwinding at the DUE
Describe Polypeptide Chain Growth
Amino acids arrive as ‘activated’ forms in ester linkage to transfer RNAs (tRNAs)
Polypeptide chain grows in the N to C direction
Handover of the growing chain to the incoming aminoacyl-tRNA
Are error rates in protein synthesis higher or lower than DNA replication?
Higher (10^-4) where as DNA replication is around (10^-9)
What does ThrRS (threonyl-tRNA synthetase) do?
Joins together the amino acid and its cognate tRNA (uses ATP -> AMP + 2Pi)
Describe the secondary and tertiary structures of tRNA
Secondary structure - single strand folded into Clover leaf structure, has an amino acid attachment site
Tertiary structure - L-shaped
Contains some modified bases
Describe the linkage of a tRNA to an amino acid
The 3’ end of the tRNA has the sequence -CCA.
Amino acid is esterified to the 3’ OH of the tRNA
What is the ‘wobble’ position?
Many degenerate codons have the first 2 bases in common, they differ in the third base at the 3’ end - this is called the wobble position.
Allows non Watson-Crick interaction at the third position
e.g. UUU codon base can pair to GAA anti codon base
What is Inosine (I)
a new base, often occurs in anticodon position 1, can recognise U, C or A at codon position 3
What do Aminoacyl-tRNA (AARSs) Synthases do?
Attach amino acids to specific tRNA molecules
Ensure faithful translation of the genetic code
Over come thermodynamic barrier to peptide bond formation
What happens when Serine is acylated instead of Threonine because they are so similar?
ThrRS is able to correct its mistakes and remove misacylated Ser-tRNA^Thr - this is called proof reading
- Editinng site selects Ser and sterically excludes Thr
Why can’t Thr be chosen over Ser?
They are so similar in shape (only one CH3 difference) so any space that Thr can fit in, so can Ser.
Describe the structure and dissociation of a ribosome.
The core of the ribosome is RNA
20 000 per cell
70S in size -> dissociates into large (50S) and small (30S) subunits
these can also be reconstituted in vitro from the purified components
How many tRNA binding sites does a ribosome have?
3 tRNA biding sites:
A site - aminoacyl
P site - peptidyl
E site - exit
Where does initiation of Protein synthesis happen?
Begins at the initiator methionine codon, AUG, fMet
- distinguish by an upsteam purine rich ribosome binding site (lots of A and G)
How is formyl Methyonine created (fMet)?
methionine is formylated after attachement to tRNA
Describe initiation of Protein Synthesis
fMet-tRNA^fMet can bind directly to the P-site, all other aa-tRNAs must first bind to the A-site
Initiation Factors (IFs) help to ensure correct order of assembly of the ribosome
GTP hydrolysis to GDP and Pi on IF-2 is a checkpoint switching mechanism
Describe Elongation of Protein Synthesis
Binding of successive aminoacyl-tRNAs to the A-site
EF-Tu bound to GTP binds to aa-tRNA and escorts it to the ribosome
- GTP hydrolysis plays proof-reading role, it checks the codon-anticodon interaction
EF-Tu-GDP dissociates from the ribosome
EF-Ts promotes exchange of. EF-Tu bound GDP for GTP to reset the cycle
Describe the peptide bond formation in elongation
Codon-anticodon interactions take place in the 30S subunit
Peptidyl transfer takes place in the 50S subunit
The amino acid/peptide in the P-site is transferred onto the amino group of the aa-tRNA in the A-site
Describe the Translocation of elogation and the enzymes involved
EF-G = translocase
it is a GTPase and promotes change in conformation of the ribosome and its movement along the mRNA
EF-G is a mimic of EF-Tu-aa-tRNA, enabling it to bind in the A-site
Describe Termination off translation of protein synthesis and how do RFs help?
Signalled by STOP codon in the A-site
Release factors (RFs) promote:
- hydrolysis of the terminal peptidyl-tRNA bond
- release of the polypeptide and the now uncharged tRNA from the P site
- dissociation of the 50S-30S complex
