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BIOM 3500 - Midterm 2: Regulation of Gene Expression > Lecture 8 - Transcription and RNA Processing > Flashcards

Lecture 8 - Transcription and RNA Processing Flashcards

1
Q

What is transcription?

A

The nucleotide sequence of the appropriate portion of the immensely long DNA molecule in a chromosome is copied into RNA

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2
Q

What is translation?

A

RNA copies of segments of the DNA that are used directly as templates to direct the synthesis of the protein

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3
Q

What is the flow of information from DNA to RNA to protein called?

A

The central dogma

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4
Q

What are some of the roles of non-coding RNA’s?

A
  • Fold into precise three-dimensional structures that have structural and catalytic roles in the cell
    Act primarily as regulators of gene expression
  • Many roles not known yet
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5
Q

Is the information present in genomes arranged in an orderly fashion?

A

No. The genes largely consist of a long string of alternating short exons and long introns. Moreover, small bits of DNA sequence that code for protein are interspersed with large blocks of seemingly meaningless DNA.

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6
Q

Why is decoding genes no simple matter?

A

Proteins that work closely with one another in the cell often have their genes located on different chromosomes, and adjacent genes typically encode proteins that have little to do with each other in the cell.

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7
Q

How does RNA differ quite dramatically in overall structure?

A

Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded, allowing it to fold up into a particular shape, just as a polypeptide chain folds up to form the final shape of the protein.

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8
Q

How is RNA in a cell made?

A

By DNA transcription

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9
Q

How does transcription begin?

A

With the opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. One of the two strands of the DNA double helix then acts as a template for the synthesis of an RNA molecule.

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10
Q

How is the nucleotide sequence of the RNA chain determined for transcription?

A

The complementary base-pairing between incoming nucleotides and the DNA template

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11
Q

Why do the RNA molecules produced by transcription released from the DNA template as single strands?

A

The RNA strand does not remain hydrogen-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms.

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12
Q

What enzymes perform transcription?

A

RNA polymerases

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13
Q

What does RNA polymerase catalyze?

A

The formation of the phosphodiester bonds that link the ribonucleotides together to form a linear chain

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14
Q

How does RNA polymerase move along the DNA?

A

Stepwise, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary base-pairing

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15
Q

What direction is the growing RNA chain being extended in?

A

5’-to-3’ direction

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16
Q

Does RNA polymerase need a primer to start an RNA chain?

A

No, because it does not need to be as accurate as DNA replication

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17
Q

Why are RNA polymerases absolutely processive?

A

The same RNA polymerase that begins an RNA molecule must finish it without dissociating from the template

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18
Q

How does RNA polymerase proofreading functino?

A

If an incorrect ribonucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that resembles the reverse of the polymerization reaction, except that a water molecule replaces the pyrophosphate and a nucleoside monophosphate is released

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19
Q

The RNA copied from the genes of DNA which specify the amino acid sequence of proteins are called what?

A

messenger RNA (mRNA)

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20
Q

What are some other common non-coding RNAs produced in cells?

A

rRNAs, tRNAs, snRNAs, snoRNAs, miRNAs, siRNAs, piRNAs, and IncRNAs

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21
Q

How does a transcription unit carrying information differ in eukaryotes and prokaryotes?

A

In eukaryotes, it is typically carrying the information for just one gene and only codes for a single RNA molecule or a single protein but in prokaryotes, the unit carries the information for several distinct proteins

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22
Q

What are the three RNA polymerases for transcription and how do they differ?

A
  • RNA polymerase I and RNA polymerase III transcribe the genes encoding tRNA, rRNA, and various small RNAs
  • RNA polymerase II transcribes most genes, including all those that encode proteins
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23
Q

In eukaryotes, what proteins do transcription initiation require?

A
  • RNA polymerase
  • General transcription factors (TFs)
  • Gene-specific transcription factors (activators and repressors)
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24
Q

What do general transcription factors do?

A
  • Help to position eukaryotic RNA polymerase correctly at the promoter
  • Aid in pulling apart the two strands of DNA to allow transcription to begin
  • Release RNA polymerase from the promoter to start its elongation mode
25
Q

What are general transcription factors “general”?

A

Because they are needed at nearly all promoters used by RNA polymerase II

26
Q

What do the general transcription factors consist of?

A

A set of interacting proteins denoted arbitrarily as TFIIA, TFIIB, TFIIC, TFIID, and so on (TFII = transcription factor for polymerase II)

27
Q

How does the assembly process of general transcription that assemble at the promoters used by RNA polymerase II begin?

A
  • The promoter contains a DNA sequences called the TATA box, which is located 30 nucleotides away from the site at which transcription is initiated
  • Through its subunit TBP (TATA binding protein), TFIID recognizes and binds the TATA box, which enables the adjacent binding of TFIIB to the BRE element, located 35 nucleotides from the site of transcription
  • The rest of the general transcription factors and RNA polymerase itself assemble at the promotor
28
Q

Once RNA polymerase II and TFIIs are brought to the promoter, what does TFIIH do?

A
  • TFIIH uses energy from ATP hydrolysis to pry apart the DNA double helix at the transcription start point, locally exposing the template strand
  • TFIIH also phosphorylates RNA polymerase II, changing the conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription
29
Q

Next, RNA polymerase II, which remains at the promoter synthesizing short lengths of RNA until what?

A

It undergoes a series of conformational changes that allow it to move away from the promoter and enter the elongation phase of transctiption

30
Q

What is the CTD?

A

A key step in the transition of RNA polymerase II, the addition of phosphate groups on the “tail” of the RNA polymerase (known as the C-terminal domain)
- Phosphorylation of CTD regulates many aspects of transcription and the processing of RNA

31
Q

What does the CTD consist of?

A

52 tandem repeats of a 7 amino acid sequence which extend from the RNA polymerase core structure

32
Q

During transcription initiation, what does TFIIH phosphorylate?

A

The serine located at the fifth position of the 7 aa sequence - Ser 5

33
Q

In a eukaryotic cell, what complex proteins does transcription initiation require on purified DNA and what do they do?

A

1) Gene regulatory proteins (transcriptional activators) must bind to specific sequences in DNA (enhancers which can be several kb away from promoter), to help attract RNA polymerase II to the start point of transcription
2) Presence of a large protein complex (mediator), which allows the activator proteins to communicate properly with the polymerase II and with the general transcription factors
3) Recruitment of chromatin-modifying enzymes, including chromatin remodelling complexes and histone-modifying enzymes

34
Q

What 4 main things are required for the function of transcription initiation?

A

TFII’s, RNA polymerase II, transcription activators, and mediator complex

35
Q

What is necessary for the movement of RNA polymerase away from the promoter (elongation)?

A

Release of all factors

36
Q

What plays a major role in factor release?

A

TFIIH

37
Q

What is TFIIH and what activity does it have?

A
  • Multicomponent protein complex
  • Helicase activity which is required for unwinding DNA at transcription start site (role of initiation)
  • Kinase activity in Cdk7 (cyclin dependent kinase-7) subunit
38
Q

What does the kinase activity of the Cdk7 subunit allow for?

A
  • Phosphorylation of Ser-5 in CTD of RNA polymerase II
    –> Causes release of most TFIIs, allowing RNA polymerase to move away from the promoter so elongation can begin
    –> Has a role in capping and splicing
  • Phosphorylates Cdk9
  • Role in cell cycle
39
Q

In addition to transcription, what other critical steps are required to produce a mature mRNA molecule?

A

1) Covalent modification of the ends of the RNA: 5’ cap (7-methyl-guanosine)
2) Removal of intron sequences hat are disregarded from the middle of the RNA transcript by the process of: RNA splicing
3) Covalent modification of the ends of the RNA: 3’ polyadenylation (poly-A-tail)

40
Q

What does modifying the eukaryotic mRNAs by capping and polyadenylation allow the cell to do?

A

Access whether both ends of an mRNA are present before it exports the RNA from the nucleus and translates it into protein

41
Q

What does RNA splicing do and what does it allow the eukaryote to do?

A

Joins different portions of a protein-coding sequence together to provide eukaryotes with the ability to synthesize several different proteins from the same gene

42
Q

What strategy greatly speeds up the overall rate of a series of consecutive reactions and how?

A

Coupling RNA processing with transcription elongation.
- RNA polymerase II in its elongation mode not only moves along the DNA synthesizing of an RNA molecule, but also processes the RNA that it produces
- Fully extended, the CTD is nearly 10x longer than the remainder of the RNA polymerase, so as aa flexible protein domain, it holds together a variety of proteins so close by that they can rapidly act when needed

43
Q

When is the new RNA molecule modified by the addition of a cap that consists of a modified guanine nucleotide?

A

As soon as RNA polymerase II has produced about 25 nucleotides of RNA

44
Q

What three enzymes act in succession to perform the capping reaction?

A

1) A phosphatase removes a phosphate from the 5’ end of the nascent RNA
2) A guanyl transferase adds a GMP in a reverse linkage (5’ to 5’ instead of 5’ to 3’)
3) Methyl transferase adds a methyl group to the guanosine

45
Q

Why are the enzymes involved in capping poised to modify the 5’ end of the nascent transcript as soon as it emerges from polymerase?

A

Because all 3 enzymes bind to the RNA polymerase tail phosphorylated at the ser-5 position (modification added by TFIIH during transcription initiation)

46
Q

How is the 5’ cap added to the transcript?

A

1) Multiple sites on CTF are gradually phosphorylated
2) Subunit of TFIIH, Cdk7, phosphorylates ser-5
–> Release of RNA polymerase II from initiation factors and allows for elongation
2) Ser-5 phosphorylated CTD recruits capping proteins (CEC)
3) CEC hops to 5’ end of transcript
4) 7-methyl guanosine cap is added to 5’ end of transcript

47
Q

What does each splicing event do?

A

Removed one intron, proceeding through two sequential phosphoryl-transfer reactions known as transesterfications which join two exons together while removing the intron between the as a “lariat”

48
Q

How is splicing carried out?

A

5) Cdk9 activated by Cdk7 phosphorylates ser-2 of CTD
6) Spliceosome is recruited because the phosphorylated ser-2 and ser-5 together serve as a recognition site for its subunits
7) Spliceosome hops onto RNA and recruits other spliceosome components
8) The U1 snRNP base pairs with the 5’ splice junction
9) Branch-point binding protein (BBP) and U2 auxiliary factor recognize the branch-point site
10) U2 snRNP displaces BBP and U2 auxiliary factor base pairs with the branch-point site consensus sequence
11) Adenine in branch-point “attacks” the 5’ junction (donor site) to form lariat
12) 5’ splice donor site attacks the 3’ junction (acceptor site) so the lariat gets released and the intron is removed

49
Q

Unlike the other steps of mRNA production, key steps in RNA splicing are performed by what?

A

RNA molecules rather than proteins
- U1, U2, U4, U5, and U6 (each associated with multiple protein subunits)

50
Q

The relatively short RNA molecules are known as what?

A

snRNAs (small nuclear RNAs)

51
Q

What is a spliceosome and what form it’s core?

A

The large assembly of RNA and protein moleules that performs pre-mRNA splicing in the cell. snRNPs form the core. (enzyme complex of snRNPs and other protein components)

52
Q

What reactions involved in splicing does the spliceosome catalyze?

A

Lariat formation and exon ligation

53
Q

What specific RNA sequences does the spliceosome recognize to catalyze intron removal?

A

-5’ splice site (donor)
-3’ splice site (acceptor)
-Branch point in the intron sequence that forms the base of the excised lariat

54
Q

What is recruited after splicing?

A

The exon junction complex (EJC)

55
Q

How is polyadenylation carried out?

A

1) Phosphorylated Ser-2 of CTD recruits CPSF and CstF which bind to AAUAAA on RNA, and promote the cleavage of RNA at a downstream CA 2) RNA is released from RNA polymerase
3) PolyA Polymerase (PAP) is recruited and adds A nucleotides to the 3’ end
4) PolyA Binding Protein (PABP) binds (has a later role in translation initiation)

56
Q

When can RNA be called mRNA?

A

Only after the 5’- and 3’-end processing and splicing have taken place

57
Q

What is the last of the RNA processing events?

A

Mature mRNAs being exported from the nucleus via a nuclear export receptor that brings it to the nuclear pore and then releases it into cytoplasm

58
Q

How does mature mRNA selectively being exported from the nucleus act as a safety mechanism?

A

Nuclear export receptor is only recruited to RNA if it is recognized by exon junction complexes (EJC) and/or 5’ cap (typically both are required for machinery to recognize it), that way RNA in the nucleus that has not been properly processed won’t escape to the cytoplasm (would stay in nucleus and eventually get degraded)

BIOM 3500 - Midterm 2: Regulation of Gene Expression (4 decks)

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