dna replication + gene expression Flashcards
what are nucleic acids
- long linear chains of RNA or DNA
- polymeric macromolecules made from nucleotide monomers
- form a structured backbone
nucleotides are made up of
1) PENTOSE SUGAR
- beta-D-ribofuranose / ribose (in RNA)
- beta-D-deoxyribofuranose / deoxyribose (in DNA)
2) NITROGENOUS BASE
- pyrimidine or purine
- attached to 1’ carbon of sugar
3) PHOSPHATE GROUP
- attached to 5’ carbon of sugar
what are dna and rna backbones based on
repeated pattern of pentose sugar linked together by phosphate group by
which bond links a phosphate to 2 sugars and at which carbons
- PHOSPHODIESTER
- 3’ carbon on one
- 5’ carbon on other
what does the formation of phosphodiester bonds mean
- nucleic acids have directionality (strand has end to end chemical orientation)
- 5’ end = free phosphate group on carbon 5’ of its terminal sugar
- 3- end = free hydroxy group on the carbon 3’ of its terminal sugar
how does dna exist in living organisms and who discovered this
double helix
watson + crick
how was the 3d structure of DNA discovered (each scientist and their discovery)
1) CHARGAFF
- determined amnt of adenine = amnt of thymine
- and cytosine = guanine
- Chargaff’s rules
2) ROSALINE + FRANKLIN
- x-ray diffraction
- discovered dna helical, 2nm diameter, complete turn of helix made every 3.4nm
3) WATSON + CRICK
- deduced dna structure from evidence from the 2 above
- proposed double helix (2 dna strands are wrapped around each other )
what is the watson-crick model
- show the chains - one in red and one in blue
- darker colour on each = the sugar phosphate backbone
- the lighter colour on each = the purine and pyrimidine bases
what did watson and crick propose about the structure of base pairs
- held together by pairing between the nitrogenous bases in the nucleotide of each opposing strand
- attraction (hydrogen bonding) holds
1) adenine and thymine together by 2 H bonds
2) cytosine and guanine together by 3 H bonds - so the dna strand is stuck together by H bonds
why is dna described as antiparallel
- because nucleic acids have directionality
- when the 2 dna strands are physically parallel they run in opposite directions (one is 3’ to 5’ the other is 5’ to 3’)
what functions does the antiparallel nature of dna serve
- make dna more structurally stable
- facilitate complementary base pairing
- enable dna strands to be held together
what is dna replication
- process of replication of entire genome prior to cell division
- copy 1 molecule of dna to produce 2 identical molecules
- essential to allow cells to divide
- requires a template and primer
what did meselson and stahl determine about the mechanism of dna replication and what does this mean
SEMI-CONSERVATIVE REPLICATION
- replication of one helix results in 2 daughter helices
- each daughter contains 1 OG parent strand and 1 newly synthesised strand
- so dna is unwound to produce 2 single stranded molecules which act as templates for the synthesis of complementary strands
what would be seen in the
2nd generation of semiconservative replication
- 4 daughter cells
- 2 with 1 strand of parent and 1 new
- 2 with 2 newly synthesised strands
how do prokaryotic cells replicate (have circular molecules of dna)
1) INITIATION
- replication begins when initiator proteins bind to a single origin of replication on the cells circular chromosome
2) ELONGATIOON
- replication proceeds around entire circuit of the chromosome in both directions from 2 replication forks
3) TERMINATION
- result = 2 dna molecules
how is the dna double helix formed
sugar phosphate backbones wind around each other
held together by hydrogen bonds
what has to be done to dna to replicate it
open it to expose the nucleotide bases (used as templates for replicating)
what steps are involved in the initiation of dna replication
1) initiator proteins unwind a short stretch of the dna double helix
2) helicase attaches to + breaks apart the hydrogen bonds between the bases on the dna strands + pulls the 2 strands apart
- > energy (from ATP hydrolysis) and specific DNA-binding proteins are also required
what does unwinding of the dna form
- separates double dna strands at origin of replication
- 2 Y shaped replication forks form
what are Y shaped replication forks
area where replication of dna will take place (actual sites where dna copied)
dna replication starts as soon as they’re established
following the formation of Y shaped replication forks, how does replication proceed
- around the entire circle of the chromosome in each direction from 2 replication forks
- results in 2 dna molecules
how does unwinding strain the molecule and how is this regulated
- causes overwinding of nearby regions
- topoisomerase enzymes
how does topoisomerase type IA regulate the overwinding of dna
1) make a cut in one strand
2) creating a gap
3) pass the other strand through the gap then seal it
4) induces positive supercoiling (relax negative supercoiled DNA), strands are separated allowing replication machinery to proceed
what does - topoisomerase action precede
replicating DNA Mechanism
which enzymes carry out dna replication and how do they work
dna polymerases
- adds nucleotides complementary to the template strands one by one to the growing dna chain
what types of polymerase have been discovered in prokaryotes and what do they do
5 types
3 main ones are type 1, 2 and 3
- types 1+3 = required for dna replication
- type 2 = involved in dna repair
what are the two main parts of the structure of dna polymerases
PART 1
- polymerase unit
- shape of a loop and right hand with a domain referred to as palm, thumb and fingers
PART 2
- domain with 3’ to 5’ exonuclease activity
- dna polymerases often contain a 3’ specific exonuclease domain
- part = believed to be for proof reading base misincorporation AND to help maintain fidelity of DNA replication
what does the “palm” part of the domain making up the right hand of part 1 of a dna polymerase do
- contains polymerase catalytic active sites where…
1) primer template junction is bound
2) incoming nucleotides are incorporated into growing primer
what does the “finger” part of the domain making up the right hand of part 1 of a dna polymerase do
role in recognition and binding of nucleotides
what does the “thumb” part of the domain making up the right hand of part 1 of a dna polymerase do
- binding of the dna substrate
- helps hold the dna in position
- helps assess accuracy of newly formed base pair in the double helix
what is used for initiating the extension of an existing dna or rna strand already paired with a template strand
RNA PRIMER (short rna fragment synthesised by primase enzyme)
1) dna polymerase starts its mechanism after primer is created + paired w a TEMPLATE dna strand
2) rna primer is removed at later stage of replication
what was previously considered the simplest mechanism for dna replication
- continuous growth of both new strands nucleotide by nucleotide at replication fork
- as replication moves from one end of dna molecule to another
- this replication is in the SAME direction
what would mechanism of dna synthesis in both directions require
bc of the antiparallel orientation of the 2 dna strands in double helix
- would require 1 daughter strand to polymerise in the 5’ to 3’ direction and the other in the 3’ to 5’ direction
what catalyses dna replication and formation of a phosphodiester bridge and what does it require
dna polymerase
- require a primer and a template
what is the process for dna synthesis
1) each incoming nucleotide forms appropriate base pair w a complimentary base in the template
2) polymerase links the incoming base w the precedent
3) dna polymerase uses energy from the hydrolysis of the phosphate bond between the free phosphates attached to each free nucleotide
4) addition of a nucleotide to growing dna strand forms a phosphodiester bond between the phosphate of the new nucleotide to the growing chain + releases 2 inorganic phosphates
dna polymerases can only add
- new nucleotides to an existing 3’ hydroxy group
- extending the 3’ end of the template chain
- SO synthesis in direction 3’ to 5’ CANNOT occur (only occurs 5’ to 3’)
how does replication occur in the bottom strand of DNA
- both strands replicated in the same time by the same rep machinery + in the same direction as the unzipping of the double strand goes along
how is the 3’ to 5’ strand synthesised in the 5’ to 3’ direction
- BOTH strands are synthesised in the 5’ to 3’ direction
- BUT
1) leading strand (complimentary to 3’ to 5’ parental dna) = synthesised continuously toward the replication fork in the direction 5’ to 3’
2) lagging strand (complimentary to 5’ to 3’ parental dna) = synthesised discontinuously in short pieces away from the replication fork
what are the pieces used to replicate the lagging strand called
Okazaki fragments (Ozaki = japanese scientist)
what primer is needed by the leading and lagging strands
leading = needs 1 primer lagging = each Okazaki fragment requires a primer to start the synthesis
summarise the process of dna replication
1) helicase unwinds dna and breaks H bonds between complementary base pairs
2) generates 2 replication forks which extend in opposite directions
3) single strand binding proteins coat the dna around the replication fork
4) following unwinding of topoisomerase binds @ region ahead of the replication forks to relax overwinding
5) primase synthesises rna primers complementary to the dna strand (1 for leading, many for lagging)
6) dna polymerase 3
7) dna polymerase 1
8) dna ligase
what does dna polymerase 3 do following the synthesis of the short rna primer
- starts adding nucleotides to the primer 3’ end
- synthesises leading + lagging strands simultaneously
- proof reads and corrects dna using 3’ exonuclease activity
how does dna polymerase 3 correct incorrect base pairing
- reverses its direction by 1 base pair of dna
- excises the incorrect base
- replaces the incorrect nucleotide
what is the function of dna polymerase 1
- remove rna primers and fill the gaps with dna in the lagging strand
what is the function of dna ligase
- seals gaps between dna fragments made by dna polymerase 1
- joins okazaki fragments together
how is there coordination between continuous synthesis of the leading and discontinuous synthesis of lagging strands
by demirisation of dna polymerase at the replication fork
1) dna polymerase moves w the rep fork to synthesise simulataneously
2) leading = dna polymerase remains attached to the DNA for continuous synthesis
3) lagging = dna polymerase elongates it in opposite direction to the fork by staying bound to the fork (initiates synthesis by rna primers)
what happens once lagging strand synthesis of one okazaki fragment is complete
newly synthesized fragment of strand loops out between the polymerase + the fork
once synthesis of the new fragment is finished - the polymerase release the loop + initiate a new fragment
DNA ligase joins the fragments into one continuous strand
how many base pairs can be added to the growing strands by polymerases per second
up to 1000
what is the primary product of transcription and what types of this exist in prokaryotes
RNA
1) ribosomal rna (rRNA)
2) transfer rna (tRNA)
3) messenger rna (mRNA)
how is RNA synthesis carried out
RNA polymerases
- prokaryotes use same one to transcribe ALL their genes
what is rna polymerase composed of in e. coli
- core enzyme
- single regulatory sub-unit (the SIGMA)
how are the core enzyme involved in rna synthesis
- comprise 4 sub-units
1) alpha
2) alpha
3) beta
4) beta prime - these assemble every time a gene is transcribed
how is the single regulatory sub-unit (the SIGMA) involved in rna synthesis
- involved only in transcription initiation
- confers transcriptional specificity so polymers begin to synthesise mRNA from an appropriate initiation site
what would the core enzyme do without sigma
transcribe from random sites
what are the processes of transcription
1) INITIATION
- RNA polymerase recognizes + binds to a promoter sequences w help of the SIGMA
2) ELONGATION
- RNA polymerase synthetises RNA complementary to the template DNA
3) TERMINATION
- RNA synthesis proceeds through terminator sequences and terminates
initiation
how does the protein responsible for transcription recognise the beginning of the element to be copied
- due to promotor sites on dna template
- promoter sequences are located just upstream of transcription start site
- promoters vary among species
initiation
where are promotor consensus sequences found (region similar across many promoters + species)
at -10 and -35 regions upstream of initiation site
initiation
in bacteria cells what is the -10 and -35 consensus sequence
- 10 region
- adenine + thymine rich (TATAAT)
- 35 region
- often TTGACA
initiation
how does rna polymerase bind to dna sequences efficiently at the transcription starting point in prokaryotes
- requires a sigma factor
- sigma subunits of prokaryotes recognise consensus seq’s @ the promoter
- once the protein dna interaction is made the subunits of the core RNA polymerase bind to the site
initiation
what happens to the sigma subunit once transcription has been initiated
dissociates from the polymerase
initiation
how does initiation of transcription begin
- binding of rna polym to the promoter
- RNA polymerase unwinds 17 base pairs of template DNA so 1 strand can be used as the template for rna synthesis
- region of unwinding = a transcription bubble
initiation
why is the -10 promoter region popular
- theyre AT rich
- lower stability of AT association (only 2 H bonds)
elongation
how does elongation of transcription begin
- with release of sigma unit from polymerase
- allows rna polymerase to proceed along the dna template synthesizing mRNA in the 5’ to 3’ direction
- ALWAYS proceeds from the template strand
elongation
what happens as elongation proceeds
- dna continuously unwound ahead of the core enzyme + rewound behind it
- rna polym proceeds along dna template adding nucleotides by base pairing w the dna template in 5’ to 3’ direction (similar to dna replication)
what is the rna product of transcription complementary to
- the template/coding strand
- EXCEPT rna contains a uracil not thymine
elongation
how is rna synthesis different to dna replication
- rna strand synthesised does not remain bound to dna template
what happens once the gene is transcribed and how can this happen
- bacteria polymerase instructed to dissociate from the dna template + liberate new rna
- 2 types of termination signal
1) rna base
2) protein based
what happens in rna based termination
- controlled by specific seq’s in dna template strand
- as polymerase nears the end of the gene being transcribed it encounters a CG rich region
- MRNA folds back on itself
- complementary CG nucleotides bind together
- result = stable hairpin/stem-loop structure followed by a series of U residues
what does the stable hairpin produced in rna based termination cause
causes polymerase to stall as soon as it begins to transcribe a region rich in AT nucleotides
how is the new mRNA transcript liberated
- complimentary UA region of mrna transcript forms only a weak interaction w template dna
- stalled polymerase
= induce enough instability for the core enzyme to break away and liberate mRNA
what is rho dependent termination
controlled by the rho protein (ATP-dependent helicase)
tracks along behind the polymerase on the growing mRNA chain
how does rho-dependent termination occur
- near the end of the gene the polymerase encounters a run of G nucleotides on the dna template + stalls
- rho protein collides with the polymerase
- rho protein binds to nascent RNA chain pulling it away from RNA polymerase and DNA template
- this interaction releases mRNA from the transcription bubble
what is mRNA used for
- template for protein synthesis via translation
what can occur simultaneously in bacteria and why
- translation often starts before transcription has finished
- because transcription occurs in the cytoplasm so transcription not physically segregated from the rest of the cell
- lack of spatial segregation means little temporal segregation for the processes
