DNA replication and gene expression

Question 1

What is the definition of DNA replication

Answer 1

the copying of one molecule of DNA to produce 2 identical molecules

Question 2

why is extreme accuracy important in DNA replication

Answer 2

to preserve the integrity of the genome for successive generations
to allow cells to divide

Question 3

How did Meselsohn and Stahl determined the semi conservative model

Answer 3

one strand of duplex is passed on unchanged to each daughter cells -‘conserved’ or template strand. The new, complementary strand of DNA is constructed based on this by the enzyme DNA polymerase

Question 4

What happens in the initiation of DNA replication

Answer 4

• replication begins at an origin of replication
• Helicase enzymes unwind DNA ( requires energy from ATP hydrolysis and specific DNA binding proteins
• Unwinding puts strain on the molecules by over- winding nearby regions

Question 5

what is Toposiomerase Typa IA repsonsible for

Answer 5

Relaxing supercoiled DNA
(Negative super coiling)

Question 6

what occurs in elongation of DNA replicaiton

Answer 6

• new strands of DNA are synthesised by DNA polymerases
• DNA replication is primed by a short stretch of RNA created by the primase enzyme. This primer is removed at a later stage of replication

Question 7

Is termination of DNA replication the same in pro/eukaryotes

Answer 7

no it is different

Question 8

how does Prokaryotic DNA replication work

Answer 8

the chromosome of a prokaryote is circular DNA molecules
Replication begins at one origin of replication and proceeds in both directs around the chromosome

Question 9

what are properties of DNA replication (6)

Answer 9

1) DNA replication is semiconservative
2) must begin at the origin of replication
3) synthesis is always in the 5’ -3’ direction
4) Synthesis can be uni or bidirectional
5) DNA replication is semi discontinuous
6) synthesis requires RNA primers

Question 10

What is the role of DNA polymerases

Answer 10

• catalyses formation of phosphodiester bridges between DNA nucleotides to form the sugar-phosphate backbone at a rate of 750 nucleotides per second
• only add nucleotides to an existing 3’ hydroxyl group
• DNA can only be synthesised in a 5’ to 3’ direction

Question 11

what is the structure of DNA polymerases

Answer 11

Have a distinctive right hand shape wroth fingers, palm and thumb.
They also contain 3’ specific exonuclease domains for proofreading base misincorporations

Question 12

Name the functions of the DNA polymerase strutcure: fingers, palm and thumb and exonuclease

Answer 12

Fingers
- lie against primer terminus
- undergo conformational change upon binding the incoming nucleotide, possible important for the catalytic mechanism

Thumb
- contacts DNA behind the primer terminus as it exists the catalytic site
- Mya play an important role in maintaining the polymerase’s hold on the primer- template duplex
- helping the polymerase synthesise DNA progressively

Palm
- The catalytic site is located in the cavity

exonuclease
- proofreads base misincorporations

Question 13

what is DNA polymerase III holoenzyme and what is its structure

Answer 13

DNA polymerase III holoenzyme is found in E.coli - enzyme combined with a coenzyme
- Made up of several accessory protein
- ring-shaped protein clamp - encircled DNA and tethers the polymerase to the duplex
- Clamp loader protein complex- assembles circular clamps around DNA
- A 3’-5’ exonuclease enables proofreading of newly synthesised DNA, correcting any errors

circular clamps- ensure processive and fast DNA replication

Question 14

when do replication forks occur

Answer 14

where 2 template strand of DNA are separated by DNA helicase
this allows for replication of each strand simultaneously

Question 15

what is the lagging strand and okazaki fragments

Answer 15

As polymerisation of a DNA nucleotide can only occur in 5’ to 3’ direction, the lagging strand (3’ to 5’) of DNA must be synthesised in short segments - called the okazaki fragments

The leading strand is synthesises continuously
hence , DNA replication is labelled as ‘semidiscontinous’

Question 16

what enables the coordination of the leadign/lagging strands

Answer 16

Dimerisation of DNA polymerase at the fork

Question 17

what initiates synthesis at the fork at an RNA primer

Answer 17

Polymerase in the lagging strand iniates synthesis
- the complementary strand elongates in direction opposite the fork by staying bound to it
-Hence the newly synthesised fragment loops out between the polymerase and the fork
- each fragment loop is released in turn

Question 18

where are enzymes for DNA replication contained within

Answer 18

replisome- large protein matrix

Question 19

what enzymes are cntained in the replisome and its functiosn

Answer 19

primosome- comprised of primase and helicase
DNA primase- makes RNA nucleotides into primers
DNA helicase- Unwinds the DNA double helix by breaking H bonds
DNA polymerase III - synthesis leading and lagging strands, can only extend from a primer, 5’-3’ polymerising activity, 3’-5’ exonucelase activity which proofreads complementary strand and corrects errors
DNA polymerase I - on lagging strand, cuts of RNA primers and fills in with DNA between okazaki fragments can proofread

Topoisomerase- alleviates positive supercoiling
RNA primer- nucleotide for the starting point of DNA replication, short strands of RNA

Single strand binding (SSB) proteins- stabilised DNA strands as they are replicated, prevents re-joining of template strands

DNA ligase- joins new complementary strand with their template strands, also joins Okazaki fragments via covalent bonds, creating ht sugar phosphate backbone on the lagging strand

Question 20

how is RNA polymerase synthesised from DNA templates

Answer 20

• iniation RNA polymerase recognises promtoer sequences
• elongation - RNA polymerase synthesises RNA complementary tot he template DNA
  (uracil in place of thymine)
• rna produce does not remain bound to the DNA template
  dsDNA is continuously unwound ahead of the core enzyme and rewound behind it
  direction of synthesies is identical to DNA synthesis - 5’-3’

TERMINATION- RNA synthesis proceeds through terminator sequences

Question 21

what are promoters

Answer 21

DNA sequences onto which the transcription machinery binds and initiated transcription. Normally exist upstream of the genes they regulate. sequence of promoters determines whether or not a gene is transcribed

Question 22

where are the 2 promoter consensus regions and what is a consensus region

Answer 22

At -10 AT rich - facilitates unwinding
at -35 bound by sigma subunits

consensus regions- regions similar across many promoters and species

Question 23

what does the sigma subunit do after transcription is iniated

Answer 23

the sigma subunit dissociated from RNA polymerase

Question 24

describe the 2 types of termination signals fro RNA synthesis

Answer 24

rho dependant termination
- rho protein
- guanine nucleotides on dna template near end of gene stalls RNA polymerase
rho protein collides with polymerase and release mRNA from trancsirpiton bubble

Rho- INdependant termination - controlled by specifc sequences in DNA template strand
GC nucleotides causes nascent mRNA to fold back on itself causing CG nucleotides to bind together
this hairpin causes polymerase to stall as it reaches AT nucelotides

Question 25

what is the importance of RNA polymerases

Answer 25

• performs transcription
• require template but NOT primer to inaite RNA synthesis

Question 26

what is the polymerase compose in E.coli

Answer 26

4 catalystic subunits and 1 regulatory subunut-sigma

4 subunits - alpha, alpha,beta,beta - make up the core enzyme

Question 27

what is the sigma subunit involved in

Answer 27

transcription iniation - confer trancriptional specificty

Question 28

What occurs in elongation

Answer 28

RNA polymerises synthesises RNA
− Transcription proceeds from the template strand
− RNA product is complementary to the template strand (and identical to the coding
strand, except for uracil in place of thymine)
− RNA polymerase proceeds along the DNA template, adding nucleotides by
complementary base pairing
− The RNA product (‘nascent RNA’) does not remain bound to the DNA template
− dsDNA is continuously unwound ahead of the core enzyme and rewound behind it
− The direction of synthesis is identical to that of DNA synthesis – 5’-3’

Question 29

when and where do alpha and beta subunits bind in DNA replication

Answer 29

2 alpha subunits- assemble polymerase on DNA strand
beta subunits - binds ribonucleoside triphosphate (NTP)
beta’ subunits binds to DNA template strand
sigma- regulatory subunit involved in transcription iniation