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IOE > dna replication + gene expression > Flashcards

dna replication + gene expression Flashcards

1
Q

what are nucleic acids

A
  • long linear chains of RNA or DNA
  • polymeric macromolecules made from nucleotide monomers
  • form a structured backbone
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2
Q

nucleotides are made up of

A

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

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3
Q

what are dna and rna backbones based on

A

repeated pattern of pentose sugar linked together by phosphate group by

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4
Q

which bond links a phosphate to 2 sugars and at which carbons

A
  • PHOSPHODIESTER
  • 3’ carbon on one
  • 5’ carbon on other
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5
Q

what does the formation of phosphodiester bonds mean

A
  • 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
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6
Q

how does dna exist in living organisms and who discovered this

A

double helix

watson + crick

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7
Q

how was the 3d structure of DNA discovered (each scientist and their discovery)

A

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 )

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8
Q

what is the watson-crick model

A
  • 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
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9
Q

what did watson and crick propose about the structure of base pairs

A
  • 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
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10
Q

why is dna described as antiparallel

A
  • 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’)

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11
Q

what functions does the antiparallel nature of dna serve

A
  • make dna more structurally stable
  • facilitate complementary base pairing
  • enable dna strands to be held together
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12
Q

what is dna replication

A
  • 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
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13
Q

what did meselson and stahl determine about the mechanism of dna replication and what does this mean

A

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
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14
Q

what would be seen in the

2nd generation of semiconservative replication

A
  • 4 daughter cells
  • 2 with 1 strand of parent and 1 new
  • 2 with 2 newly synthesised strands
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15
Q

how do prokaryotic cells replicate (have circular molecules of dna)

A

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

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16
Q

how is the dna double helix formed

A

sugar phosphate backbones wind around each other

held together by hydrogen bonds

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17
Q

what has to be done to dna to replicate it

A

open it to expose the nucleotide bases (used as templates for replicating)

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18
Q

what steps are involved in the initiation of dna replication

A

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

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19
Q

what does unwinding of the dna form

A
  • separates double dna strands at origin of replication

- 2 Y shaped replication forks form

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20
Q

what are Y shaped replication forks

A

area where replication of dna will take place (actual sites where dna copied)
dna replication starts as soon as they’re established

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21
Q

following the formation of Y shaped replication forks, how does replication proceed

A
  • around the entire circle of the chromosome in each direction from 2 replication forks
  • results in 2 dna molecules
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22
Q

how does unwinding strain the molecule and how is this regulated

A
  • causes overwinding of nearby regions

- topoisomerase enzymes

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23
Q

how does topoisomerase type IA regulate the overwinding of dna

A

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

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24
Q

what does - topoisomerase action precede

A

replicating DNA Mechanism

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25
Q

which enzymes carry out dna replication and how do they work

A

dna polymerases

- adds nucleotides complementary to the template strands one by one to the growing dna chain

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26
Q

what types of polymerase have been discovered in prokaryotes and what do they do

A

5 types
3 main ones are type 1, 2 and 3
- types 1+3 = required for dna replication
- type 2 = involved in dna repair

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27
Q

what are the two main parts of the structure of dna polymerases

A

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
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28
Q

what does the “palm” part of the domain making up the right hand of part 1 of a dna polymerase do

A
  • contains polymerase catalytic active sites where…
    1) primer template junction is bound
    2) incoming nucleotides are incorporated into growing primer
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29
Q

what does the “finger” part of the domain making up the right hand of part 1 of a dna polymerase do

A

role in recognition and binding of nucleotides

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30
Q

what does the “thumb” part of the domain making up the right hand of part 1 of a dna polymerase do

A
  • binding of the dna substrate
  • helps hold the dna in position
  • helps assess accuracy of newly formed base pair in the double helix
31
Q

what is used for initiating the extension of an existing dna or rna strand already paired with a template strand

A

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

32
Q

what was previously considered the simplest mechanism for dna replication

A
  • 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
33
Q

what would mechanism of dna synthesis in both directions require

A

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

34
Q

what catalyses dna replication and formation of a phosphodiester bridge and what does it require

A

dna polymerase

- require a primer and a template

35
Q

what is the process for dna synthesis

A

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

36
Q

dna polymerases can only add

A
  • 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’)
37
Q

how does replication occur in the bottom strand of DNA

A
  • 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
38
Q

how is the 3’ to 5’ strand synthesised in the 5’ to 3’ direction

A
  • 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
39
Q

what are the pieces used to replicate the lagging strand called

A 
Okazaki fragments 
(Ozaki = japanese scientist)
40
Q

what primer is needed by the leading and lagging strands

A 
leading = needs 1 primer
lagging = each Okazaki fragment requires a primer to start the synthesis
41
Q

summarise the process of dna replication

A

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

42
Q

what does dna polymerase 3 do following the synthesis of the short rna primer

A
  • starts adding nucleotides to the primer 3’ end
  • synthesises leading + lagging strands simultaneously
  • proof reads and corrects dna using 3’ exonuclease activity
43
Q

how does dna polymerase 3 correct incorrect base pairing

A
  • reverses its direction by 1 base pair of dna
  • excises the incorrect base
  • replaces the incorrect nucleotide
44
Q

what is the function of dna polymerase 1

A
  • remove rna primers and fill the gaps with dna in the lagging strand
45
Q

what is the function of dna ligase

A
  • seals gaps between dna fragments made by dna polymerase 1

- joins okazaki fragments together

46
Q

how is there coordination between continuous synthesis of the leading and discontinuous synthesis of lagging strands

A

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)

47
Q

what happens once lagging strand synthesis of one okazaki fragment is complete

A

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

48
Q

how many base pairs can be added to the growing strands by polymerases per second

A

up to 1000

49
Q

what is the primary product of transcription and what types of this exist in prokaryotes

A

RNA

1) ribosomal rna (rRNA)
2) transfer rna (tRNA)
3) messenger rna (mRNA)

50
Q

how is RNA synthesis carried out

A

RNA polymerases

- prokaryotes use same one to transcribe ALL their genes

51
Q

what is rna polymerase composed of in e. coli

A
  • core enzyme

- single regulatory sub-unit (the SIGMA)

52
Q

how are the core enzyme involved in rna synthesis

A
  • comprise 4 sub-units
    1) alpha
    2) alpha
    3) beta
    4) beta prime
  • these assemble every time a gene is transcribed
53
Q

how is the single regulatory sub-unit (the SIGMA) involved in rna synthesis

A
  • involved only in transcription initiation

- confers transcriptional specificity so polymers begin to synthesise mRNA from an appropriate initiation site

54
Q

what would the core enzyme do without sigma

A

transcribe from random sites

55
Q

what are the processes of transcription

A

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

56
Q

initiation

how does the protein responsible for transcription recognise the beginning of the element to be copied

A
  • due to promotor sites on dna template
  • promoter sequences are located just upstream of transcription start site
  • promoters vary among species
57
Q

initiation

where are promotor consensus sequences found (region similar across many promoters + species)

A

at -10 and -35 regions upstream of initiation site

58
Q

initiation

in bacteria cells what is the -10 and -35 consensus sequence

A
  • 10 region
  • adenine + thymine rich (TATAAT)
  • 35 region
  • often TTGACA
59
Q

initiation

how does rna polymerase bind to dna sequences efficiently at the transcription starting point in prokaryotes

A
  • 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
60
Q

initiation

what happens to the sigma subunit once transcription has been initiated

A

dissociates from the polymerase

61
Q

initiation

how does initiation of transcription begin

A
  • 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
62
Q

initiation

why is the -10 promoter region popular

A
  • theyre AT rich

- lower stability of AT association (only 2 H bonds)

63
Q

elongation

how does elongation of transcription begin

A
  • 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
64
Q

elongation

what happens as elongation proceeds

A
  • 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)
65
Q

what is the rna product of transcription complementary to

A
  • the template/coding strand

- EXCEPT rna contains a uracil not thymine

66
Q

elongation

how is rna synthesis different to dna replication

A
  • rna strand synthesised does not remain bound to dna template
67
Q

what happens once the gene is transcribed and how can this happen

A
  • bacteria polymerase instructed to dissociate from the dna template + liberate new rna
  • 2 types of termination signal
    1) rna base
    2) protein based
68
Q

what happens in rna based termination

A
  • 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
69
Q

what does the stable hairpin produced in rna based termination cause

A

causes polymerase to stall as soon as it begins to transcribe a region rich in AT nucleotides

70
Q

how is the new mRNA transcript liberated

A
  • 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

71
Q

what is rho dependent termination

A

controlled by the rho protein (ATP-dependent helicase)

tracks along behind the polymerase on the growing mRNA chain

72
Q

how does rho-dependent termination occur

A
  • 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
73
Q

what is mRNA used for

A
  • template for protein synthesis via translation
74
Q

what can occur simultaneously in bacteria and why

A
  • 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

IOE (22 decks)

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