DNA Replication

Question 1

How does DNA synthesis by DNA polymerase work?

Answer 1

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

Question 2

what is polymerase III (Pol III)?

Answer 2

Replicative polymerase

Question 3

What is the speed and error of DNA replication in E. coli?

Answer 3

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

Question 4

What allows both DNA strands to be replicated at once?

Answer 4

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

Question 5

What does DNA polymerase III require for DNA synthesis?

Answer 5

a primer

Question 6

What are the roles of DNA primase and DNA ligase

Answer 6

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

Question 7

How is DNA unwound?

Answer 7

The combined action of DnaB and SSB proteins.

Question 8

What is SSB?

Answer 8

Single-stranded DNA binding protein

Question 9

What is DnaB

Answer 9

DNA helicase

Question 10

What does the beta sliding clamp do?

Answer 10

Keeps Pol III on the DNA (t1/2 = 100 minutes)

Question 11

What is a Linking Number (Lk)?

Answer 11

The number of times one strand winds around the other.

Question 12

What is the Linking Number equation for relaxed DNA?

Answer 12

Lk^o = N/h

N = the number of base pairs

h = the helical repeat in solution = 10.5 bp/turn

σ = ΔLk/Lk^o

Question 13

What is over/under winding and how does it effect DNA replication/RNA transcription?

Answer 13

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

Question 14

How does the linking number (Lk) of parental DNA strands change after replication?

Answer 14

Reduces to zero

Question 15

How can Parental DNA strands be resolved after replication and what enzyme catalyses the process?

Answer 15

By DNA breakage and strand passage events which change the linking number (Lk) - catalysed by DNA topioisomerase

Question 16

How does DNA topoisomerase work to resolve topological complexity?

Answer 16

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

Question 17

What direction is Circular DNA replicated in?

Answer 17

bidirectionally

(also 5’ - 3’ obviously)

Question 18

How is the oriC (origin) of E. coli replication determined?

Answer 18

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

Question 19

Describe Polypeptide Chain Growth

Answer 19

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

Question 20

Are error rates in protein synthesis higher or lower than DNA replication?

Answer 20

Higher (10^-4) where as DNA replication is around (10^-9)

Question 21

What does ThrRS (threonyl-tRNA synthetase) do?

Answer 21

Joins together the amino acid and its cognate tRNA (uses ATP -> AMP + 2Pi)

Question 22

Describe the secondary and tertiary structures of tRNA

Answer 22

Secondary structure - single strand folded into Clover leaf structure, has an amino acid attachment site

Tertiary structure - L-shaped

Contains some modified bases

Question 23

Describe the linkage of a tRNA to an amino acid

Answer 23

The 3’ end of the tRNA has the sequence -CCA.

Amino acid is esterified to the 3’ OH of the tRNA

Question 24

What is the ‘wobble’ position?

Answer 24

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

Question 25

What is Inosine (I)

Answer 25

a new base, often occurs in anticodon position 1, can recognise U, C or A at codon position 3

Question 26

What do Aminoacyl-tRNA (AARSs) Synthases do?

Answer 26

Attach amino acids to specific tRNA molecules Ensure faithful translation of the genetic code Over come thermodynamic barrier to peptide bond formation

Question 27

What happens when Serine is acylated instead of Threonine because they are so similar?

Answer 27

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

Question 28

Why can’t Thr be chosen over Ser?

Answer 28

They are so similar in shape (only one CH3 difference) so any space that Thr can fit in, so can Ser.

Question 29

Describe the structure and dissociation of a ribosome.

Answer 29

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

Question 30

How many tRNA binding sites does a ribosome have?

Answer 30

3 tRNA biding sites: A site - aminoacyl P site - peptidyl E site - exit

Question 31

Where does initiation of Protein synthesis happen?

Answer 31

Begins at the initiator methionine codon, AUG, fMet - distinguish by an upsteam purine rich ribosome binding site (lots of A and G)

Question 32

How is formyl Methyonine created (fMet)?

Answer 32

methionine is formylated after attachement to tRNA

Question 33

Describe initiation of Protein Synthesis

Answer 33

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

Question 34

Describe Elongation of Protein Synthesis

Answer 34

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

Question 35

Describe the peptide bond formation in elongation

Answer 35

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

Question 36

Describe the Translocation of elogation and the enzymes involved

Answer 36

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

Question 37

Describe Termination off translation of protein synthesis and how do RFs help?

Answer 37

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