DNA replication and gene expression Flashcards
What is the definition of DNA replication
the copying of one molecule of DNA to produce 2 identical molecules
why is extreme accuracy important in DNA replication
to preserve the integrity of the genome for successive generations
to allow cells to divide
How did Meselsohn and Stahl determined the semi conservative model
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
What happens in the initiation of DNA replication
- 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
what is Toposiomerase Typa IA repsonsible for
Relaxing supercoiled DNA
(Negative super coiling)
what occurs in elongation of DNA replicaiton
- 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
Is termination of DNA replication the same in pro/eukaryotes
no it is different
how does Prokaryotic DNA replication work
the chromosome of a prokaryote is circular DNA molecules
Replication begins at one origin of replication and proceeds in both directs around the chromosome
what are properties of DNA replication (6)
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
What is the role of DNA polymerases
- 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
what is the structure of DNA polymerases
Have a distinctive right hand shape wroth fingers, palm and thumb.
They also contain 3’ specific exonuclease domains for proofreading base misincorporations
Name the functions of the DNA polymerase strutcure: fingers, palm and thumb and exonuclease
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
what is DNA polymerase III holoenzyme and what is its structure
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
when do replication forks occur
where 2 template strand of DNA are separated by DNA helicase
this allows for replication of each strand simultaneously
what is the lagging strand and okazaki fragments
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’
what enables the coordination of the leadign/lagging strands
Dimerisation of DNA polymerase at the fork
what initiates synthesis at the fork at an RNA primer
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
where are enzymes for DNA replication contained within
replisome- large protein matrix
what enzymes are cntained in the replisome and its functiosn
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
how is RNA polymerase synthesised from DNA templates
- 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
what are promoters
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
where are the 2 promoter consensus regions and what is a consensus region
At -10 AT rich - facilitates unwinding
at -35 bound by sigma subunits
consensus regions- regions similar across many promoters and species
what does the sigma subunit do after transcription is iniated
the sigma subunit dissociated from RNA polymerase
describe the 2 types of termination signals fro RNA synthesis
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
