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MolBio and Genetics I > 3. DNA replication > Flashcards

3. DNA replication Flashcards

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1
Q

What does DNA polymerase do?

A
  • uses two parental strands as a template to faithfully synthesize new daughter strands according to the specific base pairing system
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2
Q

What does DNA helicase do?

A

unwinding and strand separation using the energy relased from ATP hydrolysis

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3
Q

Why is DNA helicase so important?

A

Semi-conservative replication needs the anti-parallel parental DNA to be unwound / single stranded

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4
Q

What can hinder DNA polymerase during replication

A

secondary structures in the single strand, “hair pins”

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5
Q

What prevents secondary structure in single strands to hinder DNA polymerase?

A

single-strand binding protein monomers, this straightens region of chain

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6
Q

In which direction is the newly synthesized DNA strand?

A

5’ to 3’ direction

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7
Q

to which end are the new dNTPs added?

A

to the free 3’ OH group

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8
Q

What reaction occurs to add a new nucleotide to the DNA strand

A

condensation reaction forming a new phosphodiester bond.

  • by product: water and pyrophosphate
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9
Q

What is the Template strand?

A

the DNA complementary to the newly synthesized strand. template strand is also complimentary to primer strand

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10
Q

What does ribonucleotide reductase do?

A

helps to convert:

  • UDP -> deoxyUDP
  • ADP -> deoxy ADP etc.
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11
Q

What does kinase do with deoxyGDP/deoxyADP/etc. ?

A

converts them into dGTP/ dATP / dCTP

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12
Q

How does dTTP formed from deoxyUDP

A

deoxyUDP -> dUMP -> (using Thymidylate synthas) dTMP -> dTDP -> TTP

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13
Q

How is chromosomal DNA synthesis catalyzed

A
  • through DNA polymerase III
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14
Q

What help does DNA polymerase III need in order to bind DNA and start replication?

A
  • requires “sliding clamp” and clamp holder
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15
Q

What does the sliding clamp require

A
  • a complex of proteins (i.e. the clamp loading complex)
  • energy released from ATP hydrolysis (to load onto the DNA)
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16
Q

Can base-pairing mistakes be corrected? How?

A

Yes. by removing the incorrect base via the 3’-exonuclease activity of the DNApol III complex

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17
Q

How is DNA synthesized on the lagging strand?

A

in okazaki fragments which are eventually joined (ligated) to form a complete strand

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18
Q

How does DNA synthesis get started?

A
  • DNA pol III can only elongate onto an existing strand
  • DNA primase: a specialized RNA polymerase can start the synthesis using DNA as a template strand
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19
Q

Why can DNApol III not start DNA synthesis without a primer?

A
  • it can only elongate an already existing strand because a free 3’ OH group is required
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20
Q

What happens to the RNA primers and okazaki fragments on the lagging strand?

A
  • DNApol III finishes synthesis of okazaki fragment up until the RNA primer without joining the two molecules
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21
Q

How can the gaps between okazaki fragments and RNA primer be eliminated?

A
  • the gap is recognized by DNApol I which removes the RNA primer and fills in the space with template direct DNA
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22
Q

What does DNA ligase do?

A

It is an enzyme that joins the two adjacent DNA okazaki fragments

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23
Q

What does DNA helicase do to the replication fork?

A

at the replication fork, it causes great strain of the DNA double helix as the two ends of the helix cannot freely rotate in respect to each other

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24
Q

How are small bacterial genomes replicated?

A

they are circular and usually replicated from a single ‘replication origin’ that consists of tandem repeat rich DNA sequences

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25
Q

Do large eukaryotic genomes also have origins of replication?

A

Yes but they replicated from many different origins of replication

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26
Q

What is different about the origins of replication of eukaryotic genomes?

A
  • have multiple origins of replication compromising of many different sequence variations (approx. 100 000 in human genome)
  • not all activated at the same time
  • mechanism involves the assembly of the pre-replication complex (Pre-RC) of proteins prior to their activation and initiation of DNA synthesis
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27
Q

What is the problem with DNA replication on the lagging strand?

A

there is a progressive shortening of chromosomal ends and genetic instability, due to the removal of the RNA primer at the end of the strand.

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28
Q

What do telomers provide for the chromosomal ends?

A

telomeres provide a kind of “buffer” to protect genes located in the ‘sub telomeric’ regions

  • this way only telomeric regions are lost during DNA replication
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29
Q

What are telomeres?

A

are repetitive regions (TTAGGG) at end of chromosomes

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30
Q

How are telomers maintained?

A

telomeres are elongated by action of telomerase

31
Q

In what direction does telomerase synthesis occur?

A

3’ to 5’

32
Q

Why do telomeres have to be protected from cell’s DNA repair system?

A
  • have single stranded overhands that look like damaged DNA (due to the incomplete replication)
33
Q

How can telomeres be protected?

A

the single stranded overhangs bind to to complimentary repeats in the nearby double stranded DNA

  • this causes the telomers to form protective loops
  • proteins associated with the telomere end also help to protect them and triggering DNA repair pathways
34
Q

What does telomerase do?

A

has the ability to reverse telomere shortening

  • extends the telomeres of chromosomes
  • can make DNA using RNA as a template
35
Q

How does telomerase work?

A

the enzyme binds to a special RNA molecule

  • RNA contains a sequence that is complimentary to the telomeric repeat
  • extends the telomeric overhanging strand of telomere DNA using this complimentary RNA as a template
  • when the overhang is long enough, a matching strand can be made by normal DNA replication enzymes (eg RNA primer, DNApol III, etc)
36
Q

Describe working principle of telomerase

A
37
Q

can the mRNA of eukaryotes be immidiately translated after transcription?

A

No. There are processing and translocational steps

38
Q

Are introns or exons removed?

A

Introns are removed. Introns are non-coding regions

39
Q

What is the main difference of protein synthesis between eukaryotes and prokaryotes?

A
  • there are often polycistronic mRNA in prokaryotes
    • ​this means that lots of sequences are transcribed, which are then produce separate proteins from one mRNA
    • whereas eukaryotes have monocistronic mRNA (one mRNA codes for one protein)
40
Q

What does rRNA do?

A

ribosomal RNA forms basic structure of the ribosome and catalyze protein synthesis

41
Q

What does tRNA do?

A

transfer RNA, is central to protein synthesis as adaptors between mRNA and amino acids

42
Q

what is snRNA

A

small nuclear RNA (splicing):

  • function in a variety of nuclear processes including the splicing of pre-mRNA
43
Q

what is snoRNA

A
44
Q

what is snoRNA

A

small nucleolar RNAs used to process and chemically modify rRNAs

45
Q

what is scaRNAs

A
46
Q

what are miRNAs

A

microRNAs that regulate gene expression by blocking translation of slective mRNAs

47
Q

what are siRNA

A

small interfering RNAs turn off gene expression by directing degradation of selective mRNAs

48
Q

How does transcription start in prokaryotes?

A

(Similar to DNApol, RNA polymerase can’t bind on its own)

  • molecular interaction with the sigma factor permits RNA pol to bind to DNA at promoters (specific)
49
Q

How does the sigma factor help prokaryotic transcription?

A
  • recognizes consensus sequences in promoter
  • -35 and -10 position are reconized through sigma factor
  • positions RNA pol so that mRNA synthesis starts at +1 position
50
Q

how do variations in -35 and -10 DNA sequences affect mRNA synthesis?

A

affect how often it can initiated

51
Q

In what direction does mRNA syntheis occur?

A

5’ to 3’ direction

52
Q

is the mRNA a copy of the template/non-coding/antisense strand or the coding/sense strand?

A

mRNA is a copy of the coding/sense/non-template strand

  • mRNA is complimentary to the non-coding/anti-sense/template strand
53
Q

How does RNA pol know where to stop?

A

terminator sequences are inverted DNA repeats followed by a run of A nucleotides

  • repat copies cause hairpin loop to form (causes RNApol to stop)
54
Q

What causes RNApol to dissociate from mRNA?

A
  • weak hydrogen bonding between the run of A nucleotides in DNA strand and U ribonucleotides of mRNA cause dissociation
  • some have special helicase called RHO proteins to faciliate dissociation
55
Q

what genes does RNApol II transcribe?

A

all protein coding genes,

  • snoRNA genes
  • miRNA genes
  • siRNA genes
  • and most snRNA genes
56
Q

What does RNApol III transcribe

A
  • tRNA genes
  • 5S rRNA genes
  • some snRNA genes
  • (small RNAs)
57
Q

What else is needed other than RNApol for initiation in eukaryotic transcription

A

General tracription Factors (GTFs)

58
Q

What do GTFs do?

A
  • help RNApol recognise a T/Arich sequence in promoter called TATA box
  • correctly positions on the chromatin template to +1 position
  • called pre initiation complex
59
Q

what are specific transcription factors?

A

bind to particular DNA sequences only found in locality of certain genes

  • helps regulate which genes mRNAs are synthesised within a cell
  • may affect positively or negatively the frequency of transcription initiation (activator or inhibitor)
60
Q
A
61
Q

What are introns?

A

non-coding sequences (often very long)

62
Q

Why are the 5’ cap and 3’ polyadenylation motifs important for pre-mRNA?

A

this modification is necessary for stability and translation

63
Q

What does the enzyme capping complex (CEC) do?

A

bound to the RNApol II compley and caps the pre-mRNA cotranscriptionally

64
Q

How is the capping bonded to the eukaryotic pre-mRNA?

A

there is covalent attachment of m7G cap at the 5’ end of the pre-mRNA via an unusual 5’-5’ triphosphate bond

65
Q

What is the m7G cap important for?

A
  • mRNA is protected from degredation
  • ensures mRNA export from nucleus
  • ensures mRNA to be translated into a functional protein
66
Q

How does polydenylation of Eukaryotic Pre-mRNA occur?

A
  • CPSF and CstF recognize cleavage and polydentation signals
  • cleave free of RNApol II complex
  • polymerase (PAP) and poly-A-binding proteins (PABPs) are also recruited
  • PAP extends the 3’ end by untemplated addion of Adenosine nucleotides (then bound to PABPs
67
Q

Why is polydenylation important

A
  • participates in transcriptional termination
  • protects mRNA from degradation
  • mRNA export from nucleus
  • mRNA translation to give functional protein
68
Q

How can splicing be catalyzed?

A

through subunit containing complex called the spliceosome

69
Q

What is the subunit composition of splicosome

A

changes as the splicing of introns progresses

  • main class: snRNPs (snurps)
    • made of proteins and snRNA
70
Q

What do the snRNAs allow in snRNPs?

A
  • permit spiceosome assembly
  • recognize specific splice signals within the pre-mRNA by base pairing
71
Q

what kind of splice signals can snRNAs of snurps recognize?

A
  • 5’ splice site
  • branch site
  • 3’ splice site
72
Q

what does the recognition of the 3 splice signal sequences allow?

A

directs the correct assembly of the spliceosome and correct joining of the exons

73
Q

What is a lariat structure?

A

intron seuqnece are removed in a loop which is referred to as a lariat

74
Q

What are self splicing introns?

A

do not require help from snRNPs

  • secondary structure formation within intron caused by base pairing form inherent enzymatic activities that catalyze removal of introns and join exons
  • (do not form loops like spicosome)

MolBio and Genetics I (9 decks)

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