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Biochem > Exam 4 RNA Metabolism > Flashcards

Exam 4 RNA Metabolism Flashcards

1
Q

What are the function of ribonucleic acids (RNA) in living cells?

A

1) Messenger RNAs: encode AA sequences of all polypeptides in cell
2) Transfer RNAs: match their anticodon to mRNA while carrying specific AA used for protein synthesis
3) Ribosomal RNAs: constituents of the large and small ribosomal units

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2
Q

What are some functions that ribonucleic acids (RNA) also have in eukaryotic cells?

A
  • MicroRNAs: regulate expression of genes (via binding to specific nucleotide sequences)
  • Ribozymes: catalytic RNA molecules that act as enzymes
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3
Q

What is the function of ribonucleic acids (RNAs) in viruses?

A

It acts as genetic material in viruses

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4
Q

How is RNA made?

A

It is transcribed from DNA, and transcription is rightly regulated to control concentration of each protein

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5
Q

What are ribozymes?

A
  • Single-stranded but RNA mol. can fold into compact structures with specific functions
  • RNA molecules that are catalysts that use metal ions as cofactors (ex: group I introns)
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6
Q

What are the types of processing of mRNAs?

A
  • Splicing: eliminating introns and joining exons
  • Poly-adenylation of the 3’ end
  • Capping the 5’ end
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7
Q

How does RNA transcription in E.Coli occur?

A

-Nucleoside triphosphates add to the 3’ end of the growing RNA strand, this strand is complementary to the DNA template strand.
- Synthesis is catalyzed by RNA polymerase (enzyme) and involves 2 Mg2+ ions. – RNA polymerase unwinds 17b.p DNA and covers about 35b.p of DNA

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8
Q

What does RNA polymerase bind to? and what happens after?

A
  • RNA poly. binds to promoter to begin transcription (primer not needed)
  • Growing end of RNA base=pairs with DNA template (for 8bp and insert 50-90nt/sec), then DNA duplex unwinds and forms bubble of 17bp
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9
Q

What types of supercoils does RNA Pol generate? How are these supercoils relieved?

A

RNA Pol generates positive supercoils
- Relieved by topoisomerase

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10
Q

In terms of supercoils, what direction does transcription go?

A

Direction of transcription goes from negative to positive supercoils

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11
Q

Template strand vs. Coding strand

A
  • DNA template strand: template for RNA polymerase
  • DNA coding strand: non-template strand that has same sequence as RNA transcript (also lists the regulatory sequences)
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12
Q

Can both DNA strands code for proteins?

A

Coding or Template strand (top or bottom) can code for proteins
- Each strand codes for a # of proteins

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13
Q

What is one of the causative agents of the common cold?

A

Adenovirus
- has a linear genome

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14
Q

What is RNA polymerase?

A
  • Has 5 core subunits and a sixth subunit
  • Lacks 3’ - 5’ exonuclease so it has a high error rates
  • It binds to promoter regions to initiate transcription
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15
Q

What are the subunits in bacterial RNA polymerase?

A

Bacterial RNA Pol has at least 6 subunits
- Two a subunits: assembly and binding to UP (upstream promoter) elements
- B subunit: main catalytic subunit
- B*: DNA binding
- w subunit: protect enzyme from denaturation
- σ (sigma) subunit: directs enzyme to promoter (each class of RNA Pol has different σ subunit)

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16
Q

Common features of Promoters in E.Coli

A
  • TATA sequences: made of 2 consensus sequences -10(TATAAT) & -35(TTGACA) for σ subunit binding
  • A-T rich upstream promoter element between -40 & -60, binds the alpha subunit (and promote strand separation)
  • These sequences govern efficacy of RNA Pol binding -> affect gene expression level
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17
Q

What are nucleotides before the first nucleotide of the RNA molecule called?

A
  • Upstream nucelotides
  • Given negative values
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18
Q

What is the Footprinting technique?

A

A way to find a DNA binding site
- The DNA bound by protein will be protected from chemical cleavage at its binding site (where the protein binds, it leaves a footprint - missing bands show where RNA polymerase was bound to DNA)

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19
Q

Initiation of RNA transcription

A
  • RNA Pol binds to promoter with σ factor bound and creates a closed complex (DNA not unwounded)
  • An open complex forms where the region from -10 to +2 unwinds
  • The RNA Pol moves away from the promoter (known as clearance)
    and σ is replaced by NusA protein
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20
Q

Elongation of RNA transcription

A
  • RNA Pol binds to triphosphate nucleosides and generates RNA transcript
  • NusG binds to RNA Pol and ribsome, which links the two together. This affects the rate of transcription in prokaryotic cells
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21
Q

Process of both initiation and elongation in E.Coli

A

1) RNA Pol core binds to DNA promoter
2) Closed complex; Transcription bubble forms
3) Open complex; transcription initiated
4) Promoter clearance followed by elongation
5) Elongation continues and σ dissociates, being replaced by NusA
6) Transcription is terminated, NusA dissociates and RNA Pol is recycled

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22
Q

How is RNA transcription regulated?

A

Regulating affinity of RNA Polymerase for a promoter
- Promoter sequence: deviating from consensus sequence
- Activator proteins: CRP
- Repressor proteins: block necessary binding sites

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23
Q

Types of termination in E.Coli

A

1) p-independent
- has 3 U’s near 3’end
- Self-complementary regions from a hair pin, which makes RNA Pol and dissociate
2) p-dependent
- common CA-rich sequence known as Rut site
- p protein (helicase that binds to Rut site) processes until termination site is reached

24
Q

RNA polymerases in Eukaryotes

A
  • RNA Pol I: synthesizes pre-ribosomal RNA
  • RNA Pol II: synthesis of mRNA (fast, inhibited by a-amanitin, recognizes thousands of promoters)
  • RNA Pol III: makes tRNAs and small RNA products
  • RNA Pol IV: in plants and responsible for synthesis oof small interfering RNAs
  • Mitochondria: has own RNA Pol
25
Q

What are features of some promoters that are recognized by eukaryotic RNA Pol II?

A
  • Consensus sequence TATA
  • Inr sequence (initiator)
  • Specific regulatory sequences farther upstream
26
Q

What does eukaryotic mRNA transcription involve?

A
  • Protein-protein contacts (many transcription factors)
  • RNA pol II has 12 subunits and carboxyl-terminal domains of highly conserved repeats
27
Q

What initiates the assembly of RNA Polymerase II at Promoter? How is RNA polymerase II assembled at the Promoter?

A
  • Initiated by TATA binding protein (TBP) with the promoter (TBP is part of complex TFIID)
  • Helicase activity in TFIIH: unwinds DNA at the promotor
  • Kinase activity in TFIIH: phosphorylates the polymerase at the CTD (carboxy-terminal domain) which changes the conformation and allows RNA Pol II to transcribe
28
Q

How does RNA Elongation happen in eukaryotes?

A
  • TFIIH is released, followed by TFIIH (after 60-70nt)
  • Elongation factors bound to RNA Pol II: enhance processibity, coordinates modifications post-translation
29
Q

How does RNA Termination happen in eukaryotes?

A

Pol II is dephosphorylated

30
Q

Process of transcription in eukaryotes using RNA Pol II

A

1) Transcription factors recruit RNA pol II to DNA
2) Transcription bubble forms
3) CTD is phosphorylated during initiation and polymerase escapes the promoter
4) Elongation is aided by elongation factors after TFIIE and TFIIH dissociates
5) Elongation factors dissociate and CTD is phosphorylated. Transcription terminates facilitated by transcription factors

31
Q

What si TFIIH’s role in Nucleotide-excision repair (NER)?

A

It recruits the NER complex at a lesion

32
Q

What are TFIIH defects associated with?

A

Genetic repair diseases
- Xeroderma, Cockayne syndrome, etc

33
Q

Selective Inhibition of RNA Polymerases?

A
  • Actinomycin D and acridine: intercalate in DNA, prevents transcription
  • Rifampicin: binds to beta subunit of bacterial RNA Pols
  • a-amanitin: blocks Pol II and III of predators but doesn’t block its own Pol II
34
Q

How does the processing of mRNA happen?

A

Primary transcript = unprocessed newly synthesized RNA molecule

Processing includes:
1) Splicing out introns and rejoining exons
2) Adding a 5’-cap
3) Adding a 3’-poly(A) tail
4) Degradation

35
Q

What is the 5’-Cap added during the processing of mRNA?

A

A 7-methyl guanosine links to the 5’end via 5’,5’-triphosphate link
- Cap is formed with GTP and may include additional methylated at 2’OH (OCH3) groups of next two nucleotides following 5’ cap
- Protects RNA from nucleases and form binding site for ribosome

36
Q

Classes of Introns

A

Introns: 50-700,000 bp in length

1) Group 1, Group 2: self-splicing introns
- doesn’t need additional proteins or ATP, differs mainly in splicing mechanism

2) Spliceosomal introns: spliced by spliceosomes (same mechanism as Group 2)
- most common introns
- in protein-coding regions of euk. genomes

3) tRNA introns: spliced by protein-based enzymes
- Primary transcript cleaved by endonuclease at both intron ends
- Exons joined together by ATP-dependent ligase

37
Q

How are Group I introns spliced?

A
  • 3’OH of free guanosine is used as a nucleophile
  • Nucleophile attacks phosphodiester bond between U and A at end of intron. This releases first portion of U-ending exon
  • 3’OH of U’ending exon attacks the 5’ end of other piece of exon to rejoin the pieces
38
Q

How are group II Introns spliced?

A
  • 2’OH of an A residue in intron is used as a nucleotide
  • 2’OH attacks 5’ splice site
  • After first cleavage: second (right most) piece forms lariat-like intermediate with phosphodiester bond (formed by the A that’s activated by U2 binding near 3’ end of intron)
  • 3’OH of U’ending exon attacks 5’ end of other exon to rejoin pieces
39
Q

What is a spliceosome?

A
  • Removes spliceosome introns
  • Made of: snRNPs (small nuclear ribonuclear proteins
  • snRNA (snRNP RNA): 5 snRNAs in eukaryotes - U1, U2, U4, U5, U6)
  • GU at 5’ end, AU at 3’end: marks sites of splicing
40
Q

Assembly of Spliceosome

A

U2, U4, U5, U6 snRNP bind, bringing ~50 proteins to create spliceosome. Some parts attached to CTD of RNA Pol II, indicated coordination of splicing with transcription
- ATP need for assembly but not cleavage

41
Q

Adding the Poly(A) Tail

A
  • Serves as binding site on mRNA protecting it from degradation
  • RNA Pol II synthesizes RNA beyond cleavage signal sequence, Endonuclease cleaves RNA 10-30nt downstream to AAUAA, Polyadenylate polymerase synthesizes 80-250nt of A
42
Q

What is the cleavage signal bound by? (in addition of poly(A) tail)

A

Bound by an endonuclease and a polyadenylated polymerase that’s bound to CTD

43
Q

Calcitonin gene of rats -> CGRP (brain) and calcitonin (thyroid)

A

Primary transcript has two poly A sites: in brain and in thyroid.
- In the brain, calcitonin exon is spliced, but in the thyroid, it is retained
- This results in CGRP in the brain and calcitonin in the thyroid

44
Q

Processing of tRNA and rRNA?

A
  • Bases modified in post-transcriptional reactions
45
Q

What are MicroRNAs? What is their function in gene regulation?

A

miRNAs are short noncoding RNAs (22 nucleotides) that bind to specific regions of mRNA to alter translation. They help with cleaving mRNAs or blocking mRNA from translation

46
Q

What are the precursors that miRNAs synthesized from?

A
  • Two endoribonucleases: Drosha and Dicer
47
Q

How does RISC-miRNA prevent translation of mRNA?

A

miRNA sequence in RISC binds to complement in target mRNA
- If miRNA is “perfect” complement: target mRNA is cleaved and not translated
- If miRNA is a “partial” complement: translation is blocked

48
Q

What are ribozymes?

A

RNA molecules that catalytically cleave RNA
- cleaves themselves or another RNA
- 3D structure needed for function (denatured = inactive)
- follows Michaelis-Menton kinetics: saturable, active site, competitive inhibition, measurable Km

49
Q

Types of ribozymes

A
  • Self-splicing group I introns
  • Hammerhead ribozymes: cleaves RNA of virusoids (circular RNAs replicated by plant virus), structure looks like hammerhead
  • RNase P: cleaves precursors to tRNAs by recognizing shape of pre-tRNA and CCA sequence
50
Q

How are cellular mRNAs degraded?

A

Via ribonucleases

51
Q

What do hairpin structures in bacterial mRNA do?

A

Extend half-life

52
Q

What do the 5’cap and 3’ poly(A) tail help with in eukaryotes?

A

stability of the mRNA

53
Q

Retroviruses and synthesis

A
  • Have genomes of ssRNA
  • Has enzyme: reverse transcriptase
  • Process: virus enters host cell, reverse transcriptase makes DNA from RNA, RNA from DNA-RNA is degraded and replaced with DNA, DNA is incorporated into the host DNA
54
Q

What are the three genes and long terminal repeat found in retroviruses?

A

1) Gag (group-associated antigen): encodes long polypeptide that’s cleaved into 6 smaller proteins)
2) Pol (polypeptide): encodes protease that cleaves long poly, reverse transcriptase, and integrase to insert DNA into host
3) Env: encodes viral envelope
4) LTR (long terminal repeat): facilitates integration of virus genome into host DNA

55
Q

What are the three reactions that reverse transcriptase catalyze?

A

1) RNA-dependent DNA synthesis
2) RNA degradation
3) DNA-dependent DNA synthesis

All contain Zn2+, use a primer of tRNA, and lacks 3’-5’ proofreading, which makes reverse transcriptase error prone and high rate of mutations

56
Q

What are telomeres?

A

Structures at the ends of linear eukaryotic chromosomes
- Has repeats of T-G, A-C on opp strand
- TG strand is longer than complement and leaves 3’ overhang
- Not easily replicated using DNA polymerase (no template for RNA primer and chromosomes shorten each generation, which is fixed by adding telomeric sequences)

57
Q

Mechanism of Telomerase

A
  • Has RNA with CyAx repeat: which serves as template for synthesis of TxGy strand of telomere
  • Telomerase binds to 3’-end of chromosome and hangs off so RNA template extends beyond it
  • Telomerase extends 3’-end, using RNA of enzyme as primer
  • DNA polymerases fill in gap on bottom strand

Biochem (4 decks)

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