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MBB164 > Mitchell > Flashcards

Mitchell Flashcards

1
Q

Is RNA or DNA structure more diverse?

A
  • RNA
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2
Q

What links 2º and 3º structures of RNA?

A
  • phosphodiester bonds
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3
Q

What does the 2ºstructure of RNA typically include?

A
  • short irregular stem loops (short helices)
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4
Q

What protein is RNA bound to?

A
  • protein in ribonucleoprotein (RNP) particles
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5
Q

How is the structure of RNA stabilised?

A
  • base pairing

- base stacking interactions

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6
Q

How do RNA helices differ from DNA helices?

A
  • in RNA, major groove deeper and narrower, minor groove shallower and broader
  • coaxial stacking, adds stability = 2 short helices flipped so stacked on top of each other
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7
Q

How does base pairing occur in RNA?

A
  • U instead of T
  • involves positions 3 and 4 of pyrimidines
  • and position 1 and 6 of purines
  • contains noncanonical bps
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8
Q

What noncanonical bps can be present in RNA?

A
  • G can bp w/ U = “wobble” bp, not good or bad, can be tolerated, doesn’t affect stability
  • G can bp w/ A = “imino” bp
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9
Q

How can noncanonical bps affect the helix and when can they be important?

A
  • alter helix dimensions

- important for specific interactions w/ RNA binding proteins

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10
Q

What unusual base interactions occur w/in RNA?

A
  • noncanonical bps
  • base triplets
  • tetraloops
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11
Q

What 3º structure motifs are present in RNA?

A
  • pseudoknot structure = bping of stem loop and ss section
  • A minor motifs = nucleotides interact w/ minor grooves elsewhere in molecule, the adenine residues can fit into minor groove
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12
Q

What types of RNA are present in cells?

A
  • rRNA
  • mRNA
  • tRNA
  • snRNA (small nuclear)
  • snoRNA (small nucleolar)
  • microRNA
  • regulatory RNA
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13
Q

What RNA processing events do 1ºtranscripts undergo to yield shorter mature RNAs?

A
  • cellular RNAs processed from longer precursor molecules by endo and exonucleases
  • exoribonucleases degrade RNA from free ends of molecule
  • can have directional specificity
  • endoribonucleases cleave specific seqs or structures w/in RNA
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14
Q

What synthesises RNA during transcrip?

A
  • DNA dependent RNA pol
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15
Q

Which end of RNA are NTPs added to?

A
  • 3’ end
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16
Q

Which strand is transcribed into RNA?

A
  • template (non-coding)
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17
Q

Is joining of NTP to RNA 3’ hydroxyl reversible, and why?

A
  • basically irreversible

- by hydrolysis of pyrophosphate

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18
Q

What is a consensus seq?

A
  • most ideal seq

- those closest to it transcribed most efficiently

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19
Q

What is the structure of core RNA pol of E. Coli?

A
  • core enzyme is protein complex containing 5 subunits

- holoenzyme also contains σ factor, increasing affinity for promoters and decreasing nonspecific DNA binding

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20
Q

What does initiation of RNA transcrip involve?

A
  • promoter binding
  • DNA unwinding
  • primer synthesis
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21
Q

How is transcrip terminated in E. Coli?

A
  • intrinsic terminators –> RNA stem loop structure, G-C rich region at stem base, 3’ rich tail
  • run of Us of RNA bound to As on DNA is weakest association, so RNA pols bound can be released from DNA
  • RNA pol can also be released by rho
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22
Q

What is rho and how can it release RNA pol?

A
  • hexameric ATPase w/ ring structure

- ring can open, RNA threads through and RNA/DNA duplex destabilised by ATPase activity

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23
Q

Where is the preinitiation complex assembled? (transcrip in euks)

A
  • at pol II promoters
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24
Q

Which binding proteins bind directly to DNA in preinitiation complex? (transcrip in euks)

A
  • TATA box binding protein (TBP) in TFIID directly binds to DNA
  • after TFIIB binding, pol II binds w. TFIIF, followed by TFIIE and TFIIH
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25
Q

What is the “torpedo” model of transcrip termination?

A
  • pol II termination coupled w/ mRNA 3’ processing
  • transcript cleaved by cleavage/polyadenylation complex
  • cleavage allows degradation of downstream fragment by 5’ to 3’ exonuclease Xrn2
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26
Q

How are euk mRNAs processed in nucleus?

A
  • capping of 5’ end
  • removal of introns (splicing)
  • polyadenylation of 3’ end
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27
Q

Are transcrip and termination coupled or compartmentalised?

A
  • coupled in E. Coli

- compartmentalisation in euks, transcrip in nucleus while translation in cyto

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28
Q

How is transcrip coupled to RNA processing?

A
  • C-terminal domain (CTD) contains tandem repeats of heptapeptide
  • CTD differentially phosphorylated during transcrip
  • CTD code allows coupling of transcrip w/ RNA processing events
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29
Q

What do the terms upstream and downstream mean?

A
  • upstream = 5’ direction

- downstream = 3’ direction

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30
Q

What are the structural characteristics of euk mRNA?

A
  • monocistronic (= encode single protein)
  • ORF flanked by noncoding regions
  • 5’-5’ “cap” and “poly(A) tail” (stabilise and stimulate translation
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31
Q

What 3’ end processing do euk mRNAs undergo?

A
  • 3’ end formed by coupled cleavage and polyadenylation

- poly(A) pol adds non-templated adenylate residues to 3’ end

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32
Q

Where do cleavage and polyadenylation of mRNA occur?

A
  • 3’ of consensus seq AAUAAA
33
Q

How are protein coding genes split in euks?

A
  • euk ORF discontinuous
  • mRNAs processed from larger precursor molecules by splicing
  • introns removed
34
Q

How are intronic and exonic seqs distinguished?

A
  • through recognition of splice site seq
35
Q

Where are introns found?

A
  • usually w/in ORF
36
Q

Are intron-exon boundary seqs conserved, and why?

A
  • highly conserved

- introns need to be removed efficiently and accurately

37
Q

What is the role of the spliceosome?

A
  • carries out pre-mRNA splicing
38
Q

What do snRNPs do?

A
  • assemble and disassemble spliceosomes
39
Q

What is the catalytic mechanism of splicing?

A
  • involves 2 esterification reactions
  • not ATP dependent
  • 2’ hydroxyl group of branchpoint adenosine attacks 3’ phosphate of 5’ exon, 5’-2’ phosphodiester bond gives looped lariat
  • generated 3’ hydroxyl group attacks 5’ phosphate of 3’ exon, releasing the lariat
40
Q

What are RNA enzymes known as?

A
  • ribozymes
41
Q

What is nuclear pre-mRNA splicing thought to have evolved from?

A
  • self splicing introns
42
Q

How many diff reading frames does coding seq have?

A
  • 3
43
Q

What does it mean to say the genetic code is non-punctuated?

A
  • no gaps between codons
44
Q

What is the initiation codon, and what does it encode?

A
  • AUG

- encodes Met

45
Q

What are the stop codons?

A
  • TAA
  • TAG
  • TGA
46
Q

What is the Shine-Dalgarno (SD) seq?

A
  • seq w/in mRNA recognised by bping w/ nucleotides at 3’ end
47
Q

How was genetic code cracked by in vitro translation?

A
  • isolate E. Coli cell extract
  • degrade mRNA w/ RNase
  • inactivate RNase
  • add RNA and AAs
  • ppt protein w/ TCA and collect by filter binding
  • detect whether radiolabelled AA incorporated
48
Q

How was genetic code cracked by ribosome binding?

A
  • aminoacyl-tRNA binds to ribosome in presence of its cognate codon
  • filter binding assays used to test for complex formation of ribosome/tRNA/codon complexes
49
Q

What are synonymous codons?

A
  • encode same AA

- tend to differ at 3rd nucleotide

50
Q

What is codon bias?

A
  • synonymous codons used to variable extents
51
Q

Why is noncanonical bping at 3rd base of codons sometimes necessary?

A
  • not all organisms have 61 diff tRNAs
52
Q

What happens during noncanonical bping at 3rd base of codons?

A
  • tRNA can bp w/ more than 1 codon, due to “wobble”
  • many tRNAs have mod nucleotide at 1st position of anticodon
  • G in 1st position can recognise codons ending w/ C or U
  • inosine bp w/ A, C or U
  • tRNA Leu (IAG) can recognise CUA, CUC, CUU and UUA codons
53
Q

What are the 21st and 22nd AAs?

A
  • 21st = selenocysteine, selenium essential for many organism (inc humans), selenoproteins synthesised by incorporating seloncysteine, UGA sometimes decoded by tRNA Sec
  • 22nd = pyrrolysine, encoded by UAG in some archaeabacteria
54
Q

How are tRNAs generated?

A
  • gen from large precursors
  • RNase P gen 5’ end
  • 3’ end gen by endo and/or exonucleases
  • tRNA nucleotidyltransferase adds CCA to 3’ end
  • tRNA splicing distinct from pre-mRNA splicing
55
Q

How is tRNAs cloverleaf 2º structure achieved?

A
  • 5’ and 3’ ends drawn together
  • AA attached to 3’ hydroxyl group of 3’ AA
  • specific nucleotides w/in tRNAs post transcriptionally modified
  • mod of 1st position of anticodon allows wobble
56
Q

How are tRNAs folded into an L shape?

A
  • coaxial stacking of helices and bping between ends of TψC and D loops prod flat L shaped molecule
  • anticodon loop and aminoacyl group positioned at opp ends of molecule
57
Q

How are tRNAs charged?

A
  • “charged” w/ approp AA by aminoacyl-tRNA synthetases
  • single enzyme charges all isoacceptor tRNAs
  • reaction req ATP
  • AA linked to tRNA by ester linkage between CA groups and 3’ OH of terminal molecule
58
Q

What is the overall reaction for tRNAs becoming charged?

A
  • tRNA + AA –> AA-tRNA
59
Q

How are tRNAs identified? (“2nd genetic code”)

A
  • aminoacyl-tRNA synthetases recognise all isoacceptor tRNAs and distinguish them from noncognate tRNAs
  • tRNA recognition involves identity and -ve identity elements
60
Q

How does kinetic proofreading of tRNA charging occur?

A
  • AA + ATP –> AA-AMP + PPi
  • tRNA + AA-AMP –> AA-tRNA + AMP
  • substrate binding alone doesn’t provide enough specificity, so coupled to tRNA aminoacylation by ATP hydrolysis
61
Q

What subunits do ribosomes consist of and what happens at each subunit?

A
  • 2 unequal subunits
  • each subunit contains large RNA molecule and ~20-50 unique proteins
  • codon/anticodon binding occurs on small subunit
  • peptide bond formation occurs on large subunit
62
Q

How does ribosome structure reflect rRNA folding?

A
  • bacterial and euk ribosomes have similar morphologies
  • conserved and modified resides w/in rRNA map to functional sites w/in ribosome
  • peptide bond formation is RNA catalysed reaction
  • ribosomes have 3 tRNA binding sites
63
Q

What does ribosome synthesis req?

A
  • 100s of proteins and snoRNAs
64
Q

What are rRNAs processed from?

A
  • large pol I transcript
65
Q

Is organisation of rRNA genes conserved?

A
  • yes
66
Q

Why is peptidyltransfer the essence of translation?

A
  • anticodons and aminoacyl groups of tRNA molecules lie close together
  • peptide bond formation (peptidyltransfer reaction) involves nucleophilic attack of aminoacyl-tRNA on carboxyl group of peptidyl-tRNA
67
Q

What are the 3 ribosomal tRNA binding sites? (translation elongation cycle)

A
  • A (aminoacyl) site
  • P (peptidyl) site
  • E (exit) site
68
Q

How many tRNAs are bound in translation elongation cycle at any 1 time?

A
  • 2

- P and E sites or A and P sites

69
Q

How are GTPases involved in translation elongation

A
  • AA-tRNA brought to ribosome by elongation factor EF-Tu (or EE1A in euks)
  • translocation req another elongation factor, EFG (EF2 in euks)
  • these elongation factors are GTPases
  • 2 GTP molecules hydrolysed per incorporated AA
70
Q

How does 16S/mRNA bping slect initiation codons?

A
  • SD seq w/in mRNA recognised by bping w/ nucleotides at 3’ end of 16S rRNA
  • SD/anti-SD interaction positions initiation codon in ribosomal P-site
71
Q

How does the initiator tRNA bind to initiation codon?

A
  • initiation codon lies in P site
  • cells contain 2 distinct Met-tRNAs
  • initiator tRNAs used to recognise initiation codon
  • recognised internal AUG
72
Q

How do initiator tRNAs vary in proks and euks?

A
  • proks = tRNA met f, methionyl group formylated (addition of C=O bonded to H)
  • euks = tRNA met, and isn’t modified
73
Q

What cotranslationally modifies most euk proteins?

A
  • N terminal acetyltransferase
74
Q

How is translation initiated in E. Coli?

A
  • tRNA met f binds p site
  • bound by initiation factor IF2 (GTPase) in ternary complex
  • after formation of 30S/mRNA/tRNA complex, 50S subunit associates
  • GTP hydrolysis by IF2 then allows formation of initiation complex
75
Q

How is translation initiated in euks?

A
  • also involves formation of ternary complex, which binds 40S and other factors
  • 40S/eIF2/tRNA complex then binds 5’ end of mRNA through interactions w/ cyto cap binding complex, eIF4F
  • 40S/eIF2/tRNA/eIF4F complex scans along mRNA using helicase activity of IF4F until finds AUG w/in approp context
  • GTP hydrolysis by eIF2, followed by release of initiation factors and 60S binding
  • binding of 60S req another GTPase, eIF5
76
Q

How is translation terminated?

A
  • stop codons by protein termination factors, not tRNAs
  • binding of release factor (RF1 or RF2, eRF1 in euks) triggers peptide hydrolysis
  • RF3 (or eRF3) is GTPase, allows release of RF1 or Rf2 from ribosome
  • set of factors inc EF-G dissociate ribosome after termination
  • various other protein factors req to remove remaining tRNA molecule in P-site
77
Q

What do many widely used antibiotics target?

A
  • proks ribosome
78
Q

How can toxins affect ribosome?

A
  • Diphtheria toxin encoded by phage in C. diphtheriae
  • toxin transfers ADP-ribosyl group from NAD+ to modified His residue in EF2
  • sarcin/ricin loop bound by EF2
  • nicin and related plant lectins are N-glycosidases that depurinate A4324, blocking EF2 binding

MBB164 (7 decks)

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