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
Do large eukaryotic genomes also have origins of replication?
Yes but they replicated from many different origins of replication
26
What is different about the origins of replication of eukaryotic genomes?
* 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
27
What is the problem with DNA replication on the lagging strand?
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.
28
What do telomers provide for the chromosomal ends?
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
29
What are telomeres?
are repetitive regions (TTAGGG) at end of chromosomes
30
How are telomers maintained?
telomeres are elongated by action of telomerase
31
In what direction does telomerase synthesis occur?
3' to 5'
32
Why do telomeres have to be protected from cell's DNA repair system?
* have single stranded overhands that look like damaged DNA (due to the incomplete replication)
33
How can telomeres be protected?
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
What does telomerase do?
has the ability to reverse telomere shortening * extends the telomeres of chromosomes * can make DNA using RNA as a template
35
How does telomerase work?
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
Describe working principle of telomerase
37
can the mRNA of eukaryotes be immidiately translated after transcription?
No. There are processing and translocational steps
38
Are introns or exons removed?
Introns are removed. Introns are non-coding regions
39
What is the main difference of protein synthesis between eukaryotes and prokaryotes?
* 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
What does rRNA do?
*ribosomal RNA* forms basic structure of the ribosome and catalyze protein synthesis
41
What does tRNA do?
transfer RNA, is central to **protein synthesis** as adaptors between mRNA and amino acids
42
what is **snRNA**
small nuclear RNA (splicing): * function in a variety of nuclear processes including the splicing of pre-mRNA
43
what is snoRNA
44
what is snoRNA
small nucleolar RNAs used to process and chemically modify rRNAs
45
what is scaRNAs
46
what are miRNAs
microRNAs that regulate gene expression by blocking translation of slective mRNAs
47
what are siRNA
small interfering RNAs turn off gene expression by directing degradation of selective mRNAs
48
How does transcription start in prokaryotes?
(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
How does the sigma factor help prokaryotic transcription?
* 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
how do variations in -35 and -10 DNA sequences affect mRNA synthesis?
affect how often it can initiated
51
In what direction does mRNA syntheis occur?
5' to 3' direction
52
is the mRNA a copy of the template/non-coding/antisense strand or the coding/sense strand?
mRNA is a **copy** of the coding/sense/non-template strand * mRNA is **complimentary** to the non-coding/anti-sense/template strand
53
How does RNA pol know where to stop?
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
What causes RNApol to dissociate from mRNA?
* 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
what genes does RNApol II transcribe?
all protein coding genes, * snoRNA genes * miRNA genes * siRNA genes * and most snRNA genes
56
What does RNApol III transcribe
* tRNA genes * 5S rRNA genes * some snRNA genes * (small RNAs)
57
What else is needed other than RNApol for initiation in eukaryotic transcription
General tracription Factors (GTFs)
58
What do GTFs do?
* 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
what are specific transcription factors?
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
61
What are introns?
non-coding sequences (often very long)
62
Why are the 5' cap and 3' polyadenylation motifs important for pre-mRNA?
this modification is necessary for stability and translation
63
What does the enzyme capping complex (CEC) do?
bound to the RNApol II compley and caps the pre-mRNA cotranscriptionally
64
How is the capping bonded to the eukaryotic pre-mRNA?
there is covalent attachment of m7G cap at the 5' end of the pre-mRNA via an unusual **5'-5' triphosphate bond**
65
What is the m7G cap important for?
* mRNA is protected from degredation * ensures mRNA export from nucleus * ensures mRNA to be translated into a functional protein
66
How does polydenylation of Eukaryotic Pre-mRNA occur?
* 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
Why is polydenylation important
* participates in transcriptional termination * protects mRNA from degradation * mRNA export from nucleus * mRNA translation to give functional protein
68
How can splicing be catalyzed?
through subunit containing complex called the **spliceosome**
69
What is the subunit composition of splicosome
changes as the splicing of introns progresses * main class: **snRNPs (**snurps) * made of proteins and snRNA
70
What do the snRNAs allow in snRNPs?
* permit spiceosome assembly * recognize **specific splice signals** within the pre-mRNA by base pairing
71
what kind of splice signals can snRNAs of snurps recognize?
* 5' splice site * branch site * 3' splice site
72
what does the recognition of the 3 splice signal sequences allow?
directs the correct assembly of the spliceosome and correct joining of the exons
73
What is a lariat structure?
intron seuqnece are removed in a loop which is referred to as a lariat
74
What are self splicing introns?
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)
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