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MBB164 - Molecular Biology > The Mechanisms of Translation and Transcription > Flashcards

The Mechanisms of Translation and Transcription Flashcards

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1
Q

What carriers out transcription?

A

DNA-dependent RNA polymerases (RNAPs)

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2
Q

Between which sites does transcription occur?

A

specific “start” and “stop” sites

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3
Q

At what part of the polynucleotide chain are nucleotides added?

A

the 3’ end of the polynucleotide chain

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4
Q

What synthesises RNA?

A

DNA-dependent RNA polymerases (RNAPs)

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5
Q

In which strand of the DNA is the genetic information transcribed into RNA?

A

the coding/sense strand

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6
Q

What does NTP stand for?

A

nucleotide triphosphate

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7
Q

How are NTPs selected for?

A

by base-pairing with the template strand

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8
Q

At which end of the extending RNA strand are NTPs added?

A

at the 3’ end

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9
Q

What does the RNA Polymerase active site contain?

A

a short RNA/DNA heteroduplex

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10
Q

What is transcription?

A

The process by which genetic sequence information that is stored in double-stranded DNA is copied into single-stranded RNA molecules

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11
Q

What do RNA polymerases do?

A
  • join nucleotides together in a specific order

- determined by the sequence of the DNA

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12
Q

What is the process of transcription intrinsically dependent upon?

A

The DNA template

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13
Q

Where is transcription initiated?

A

specific transcription start sites

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14
Q

Where is transcription terminated?

A

specific transcription termination sites

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15
Q

In what direction is the RNA polynucleotide generated?

A

5’ to 3’

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16
Q

What must occur for complementary base-pairing to happen between DNA and RNA nucleotides?

A

a region of double-stranded DNA found within the active site of the DNA-dependent RNA polymerase is melted into constitutive single strands

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17
Q

How long is the transcription bubble?

A

approximately 10 nucleotides long

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18
Q

What does transcription produce?

A

an RNA transcript with the same genetic information sequence as the coding or sense strand of DNA

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19
Q

What type of chemical reaction occurs via the addition of a nucleotide to the RNA transcript?

A

a nucleophilic substitution reaction

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20
Q

What is the basic mechanism of this nucleophilic substitution reaction?

A
  • 3’ OH group of the RNA attacks the alpha-phosphate group of the incoming NTP
  • the oxygen atom connecting the alpha and the beta phosphate groups is displaced
  • pyrophosphate (diphosphate ester) is released
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21
Q

How is Pyrophosphate/diphosphate ester abbreviated?

A

PPi

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22
Q

How does the 3’ OH group act as a nucleophile?

A
  • has a lone pair of electrons

- giving it a partially negative charge

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23
Q

How does the alpha-phosphate of the NTP act as an electrophile?

A
  • has a partially positive charge

- due to neighbouring electronegative oxygen atoms

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24
Q

In what direction are nucleotide sequences always written?

A

in the 5’ - > 3’ direction

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25
Q

What does the coding strand encode for?

A

encodes for the functional protein product of the gene

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26
Q

Are the template and coding strands gene-specific?

A
  • yes

- it is only specific to that particular transcription “unit”

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27
Q

In what direction(s) can two RNA polymerases move along DNA?

A
  • convergently (the RNA polymerases come closer together)

- divergently (RNA polymerases move away from each other)

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28
Q

Which sequence do we refer to when talking about the DNA sequence of a gene?

A

the coding strand’s genetic sequence

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29
Q

How does transcription termination occur in prokaryotic cells?

A

via destabilising the RNA/DNA heteroduplex

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30
Q

Which enzymes in prokaryotes can synthesise RNA in the absence of DNA?

A
  • polynucleotide phosphorylase
  • nucleotidyltransferase
  • poly(A) polymerase
31
Q

What is the E. coli RNA polymerase core enzyme?

A
  • a protein complex containing 5 subunits

- a,a,ß,ß’,w

32
Q

What do the beta subunits do in prokaryotic RNA polymerase?

A
  • ß and ß’ have a catalytic function
  • groove between the subunits allows for the binding of the double-stranded DNA
  • where RNA synthesis takes place
33
Q

What do the alpha subunits do in prokaryotic RNA polymerase?

A
  • a and a bind transcription factors

- which regulates the enzymes activity

34
Q

what does the omega subunit do in the prokaryotic RNA polymerase?

A

w is involved in the assembly and stability of the prokaryotic RNA polymerase

35
Q

How are prokaryotic RNA polymerases targeted to transcription sites?

A
  • via specific DNA-binding proteins

- sigma factors

36
Q

What does the enzyme polynucleotide phosphorylase (PNPase) do?

A
  • synthesise RNA
  • requires nucleoside diphosphate (DNPs)
  • functions equally well in the reverse reaction to degrade RNA
37
Q

What do tRNA nucleotidyltranserases do?

A
  • add a small number of specific nucleotides onto the 3’ end of tRNAs
  • without a DNA template
  • ensure tRNA molecules have the correct trinucleotide sequence at their 3’ end
38
Q

What does poly(A) polymerase (PAP) do?

A
  • extended 3’ ends of mRNA molecules with a stretch of A residues
  • in the absence of a DNA template sequence
39
Q

What is a general feature of gene expression in both prokaryotic and eukaryotic organisms?

A

the ability of particular proteins to recognise and specifically interact with defined nucleotide sequences within DNA or RNA

40
Q

What does the protein sigma factor do?

A
  • specifically recognises the sequence of NDA at the 5’ end of the gene
  • so that RNA polymerase is correctly positioned to initiate transcription at the correct site
41
Q

What is the region of DNA which RNA binds to to start transcription called?

A

the promoter region

42
Q

How does the polymerase/sigma factor complex initially bind to the DNA?

A
  • weakly and non-specifically

- so it can scan along the length of the DNA until it interacts with a gene promoter sequence

43
Q

What happens when the RNA polymerase binds to the promoter gene?

A
  • the RNA polymerase adopts an open and catalytically active conformation
44
Q

What forms/happens once the RNA polymerase has switched to an open conformation?

A
  • transcription bubble in the DNA binding site via the double-stranded DNA melting
  • the enzyme synthesises a short RNA primer
45
Q

When is the sigma factor released?

A

once the RNA/DNA heteroduplex is of sufficient length

46
Q

What happens once the sigma factor is released?

A
  • RNA polymerase moves away from the promoter

- RNA polymerase engages fully int he process of transcription

47
Q

How is RNA polymerase different from DNA polymerase?

A

RNA polymerase has inherent DNA unwinding and primate activity

48
Q

What is the stable interaction between the RNAP and DNA dependent on?

A

the DNA/RNA heteroduplex within the enzymes active site

49
Q

What does destabilisation of the heteroduplex result in?

A
  • the release of RNA polymerase

- transcription termination

50
Q

What would continued transcription result in?

A
  • block transcription events of other genes
  • generate unwanted RNA sequences that would titrate out RNA binding proteins
  • wasteful of important nucleotide resources
51
Q

What is the least stable heteroduplex sequence?

A

A residues in DNA and U residues in RNA

52
Q

What will a continuous stretch of A residues in the DNA template strand result in?

A
  • a continuous stand of U residues in RNA
  • this continuously unstable heteroduplex will destabilise the DNA/RNA interaction
  • cause release of the RNA polymerase
53
Q

What methods can be used in prokaryotes to terminate transcription?

A
  • A-rich template strand DNA sequence

- Rho factor helices activity

54
Q

What is Rho?

A
  • a termination factor

- an RNA helices

55
Q

How does Rho work?

A
  • triggers the release of RNA polymerase

- uses the energy released through ATP hydrolysis to drive dissociation of the polymerase from the DNA

56
Q

What are the 3 nuclear RNA polymerases present in eukaryotic cells?

A
  • RNA polymerase I
  • RNA polymerase II
  • RNA polymerase III
57
Q

What does RNA polymerase I synthesise?

A
  • rRNA

- single transcript which is later process into 3 of the 4 rRNAs

58
Q

What does RNA polymerase II synthesise?

A
  • mRNA

- noncoding RNAs

59
Q

What does RNA polymerase III synthesise?

A
  • tRNA

- 5S rRNA

60
Q

What do the 3 eukaryotic RNA polymerases have in common?

A
  • common subunits

- a conserved structure which is homologous to prokaryotic RNA polymerase

61
Q

What do the 3 eukaryotic RNA polymerases not have in common?

A
  • unique subunits
  • all 3 are visually different
  • all 3 interact with distinct sets of genes
62
Q

How are the genes within mitochondria and chloroplasts transcribed?

A

by additional RNA polymerases found within these organelles

63
Q

How are eukaryotic RNA polymerases assembled at the promoter?

A

by protein-complexes / general transcription factors

64
Q

How is eukaryotic RNA polymerase II assembly at the promoter initiated?

A
  • transcription factor IID (TFIID) binds to an A/T rich sequence in the gene promoter region
  • known as the TATA box
  • through the TFIID subunit called TATA-box binding protein (TBP)
65
Q

How does the TATA-box binding protein interact with the DNA?

A
  • binds to the minor groove of the double-stranded DNA
66
Q

What does the binding of the TATA-box binding protein to the DNA cause?

A
  • the DNA to bend

- allowing the recruitment of further transcription factors

67
Q

After more transcription factors are recruited to the TATA-box binding protein is bound what happens?

A
  • preinitiation complex (PIC) is assembled at the promoter

- involving RNAPII and a number of general transcription factor complexes (gTFs)

68
Q

Where does RNAPIII termination occur?

A
  • at dA tracts in the coding strand

- resulting in transcripts having oligo (U) sequences at their 3’ end

69
Q

How does termination of transcription by RNAPII occur?

A
  • destabilisation of the polymerase/DNA complex
  • through the action of exoribonucleases
    the cleavage and polyadenylation complex endonucleolytically cuts the transcript and adds a poly(A) tail
70
Q

What does the poly(A) tail of the RNA transcript allow for?

A
  • binding of specific nuclear proteins which protect the released fragment from degradation
71
Q

What happens to the 3’ fragment generated by cleavage?

A
  • it contains an accessible 5’ end
  • targeted by exoribonucleases which degrade downstream
  • until the RNA polymerase is reached
  • this displaces it from the DNA
72
Q

In prokaryotes is translation and transcription couples?

A
  • yes
  • mRNA is translated into protein whilst it is being made by the RNAP
  • allows for rapid gene expression
73
Q

In eukaryotes is translation and transcription couples?

A
  • no
  • transcription occurs in the nucleus
  • translation occurs in the cytoplasm
  • allows for regulation at different steps in the gene expression pathway

MBB164 - Molecular Biology (10 decks)

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