Mitchell Flashcards
Is RNA or DNA structure more diverse?
- RNA
What links 2º and 3º structures of RNA?
- phosphodiester bonds
What does the 2ºstructure of RNA typically include?
- short irregular stem loops (short helices)
What protein is RNA bound to?
- protein in ribonucleoprotein (RNP) particles
How is the structure of RNA stabilised?
- base pairing
- base stacking interactions
How do RNA helices differ from DNA helices?
- 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
How does base pairing occur in RNA?
- U instead of T
- involves positions 3 and 4 of pyrimidines
- and position 1 and 6 of purines
- contains noncanonical bps
What noncanonical bps can be present in RNA?
- 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
How can noncanonical bps affect the helix and when can they be important?
- alter helix dimensions
- important for specific interactions w/ RNA binding proteins
What unusual base interactions occur w/in RNA?
- noncanonical bps
- base triplets
- tetraloops
What 3º structure motifs are present in RNA?
- 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
What types of RNA are present in cells?
- rRNA
- mRNA
- tRNA
- snRNA (small nuclear)
- snoRNA (small nucleolar)
- microRNA
- regulatory RNA
What RNA processing events do 1ºtranscripts undergo to yield shorter mature RNAs?
- 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
What synthesises RNA during transcrip?
- DNA dependent RNA pol
Which end of RNA are NTPs added to?
- 3’ end
Which strand is transcribed into RNA?
- template (non-coding)
Is joining of NTP to RNA 3’ hydroxyl reversible, and why?
- basically irreversible
- by hydrolysis of pyrophosphate
What is a consensus seq?
- most ideal seq
- those closest to it transcribed most efficiently
What is the structure of core RNA pol of E. Coli?
- core enzyme is protein complex containing 5 subunits
- holoenzyme also contains σ factor, increasing affinity for promoters and decreasing nonspecific DNA binding
What does initiation of RNA transcrip involve?
- promoter binding
- DNA unwinding
- primer synthesis
How is transcrip terminated in E. Coli?
- 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
What is rho and how can it release RNA pol?
- hexameric ATPase w/ ring structure
- ring can open, RNA threads through and RNA/DNA duplex destabilised by ATPase activity
Where is the preinitiation complex assembled? (transcrip in euks)
- at pol II promoters
Which binding proteins bind directly to DNA in preinitiation complex? (transcrip in euks)
- TATA box binding protein (TBP) in TFIID directly binds to DNA
- after TFIIB binding, pol II binds w. TFIIF, followed by TFIIE and TFIIH
What is the “torpedo” model of transcrip termination?
- 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
How are euk mRNAs processed in nucleus?
- capping of 5’ end
- removal of introns (splicing)
- polyadenylation of 3’ end
Are transcrip and termination coupled or compartmentalised?
- coupled in E. Coli
- compartmentalisation in euks, transcrip in nucleus while translation in cyto
How is transcrip coupled to RNA processing?
- C-terminal domain (CTD) contains tandem repeats of heptapeptide
- CTD differentially phosphorylated during transcrip
- CTD code allows coupling of transcrip w/ RNA processing events
What do the terms upstream and downstream mean?
- upstream = 5’ direction
- downstream = 3’ direction
What are the structural characteristics of euk mRNA?
- monocistronic (= encode single protein)
- ORF flanked by noncoding regions
- 5’-5’ “cap” and “poly(A) tail” (stabilise and stimulate translation
What 3’ end processing do euk mRNAs undergo?
- 3’ end formed by coupled cleavage and polyadenylation
- poly(A) pol adds non-templated adenylate residues to 3’ end
Where do cleavage and polyadenylation of mRNA occur?
- 3’ of consensus seq AAUAAA
How are protein coding genes split in euks?
- euk ORF discontinuous
- mRNAs processed from larger precursor molecules by splicing
- introns removed
How are intronic and exonic seqs distinguished?
- through recognition of splice site seq
Where are introns found?
- usually w/in ORF
Are intron-exon boundary seqs conserved, and why?
- highly conserved
- introns need to be removed efficiently and accurately
What is the role of the spliceosome?
- carries out pre-mRNA splicing
What do snRNPs do?
- assemble and disassemble spliceosomes
What is the catalytic mechanism of splicing?
- 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
What are RNA enzymes known as?
- ribozymes
What is nuclear pre-mRNA splicing thought to have evolved from?
- self splicing introns
How many diff reading frames does coding seq have?
- 3
What does it mean to say the genetic code is non-punctuated?
- no gaps between codons
What is the initiation codon, and what does it encode?
- AUG
- encodes Met
What are the stop codons?
- TAA
- TAG
- TGA
What is the Shine-Dalgarno (SD) seq?
- seq w/in mRNA recognised by bping w/ nucleotides at 3’ end
How was genetic code cracked by in vitro translation?
- 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
How was genetic code cracked by ribosome binding?
- 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
What are synonymous codons?
- encode same AA
- tend to differ at 3rd nucleotide
What is codon bias?
- synonymous codons used to variable extents
Why is noncanonical bping at 3rd base of codons sometimes necessary?
- not all organisms have 61 diff tRNAs
What happens during noncanonical bping at 3rd base of codons?
- 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
What are the 21st and 22nd AAs?
- 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
How are tRNAs generated?
- 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
How is tRNAs cloverleaf 2º structure achieved?
- 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
How are tRNAs folded into an L shape?
- 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
How are tRNAs charged?
- “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
What is the overall reaction for tRNAs becoming charged?
- tRNA + AA –> AA-tRNA
How are tRNAs identified? (“2nd genetic code”)
- aminoacyl-tRNA synthetases recognise all isoacceptor tRNAs and distinguish them from noncognate tRNAs
- tRNA recognition involves identity and -ve identity elements
How does kinetic proofreading of tRNA charging occur?
- 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
What subunits do ribosomes consist of and what happens at each subunit?
- 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
How does ribosome structure reflect rRNA folding?
- 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
What does ribosome synthesis req?
- 100s of proteins and snoRNAs
What are rRNAs processed from?
- large pol I transcript
Is organisation of rRNA genes conserved?
- yes
Why is peptidyltransfer the essence of translation?
- 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
What are the 3 ribosomal tRNA binding sites? (translation elongation cycle)
- A (aminoacyl) site
- P (peptidyl) site
- E (exit) site
How many tRNAs are bound in translation elongation cycle at any 1 time?
- 2
- P and E sites or A and P sites
How are GTPases involved in translation elongation
- 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
How does 16S/mRNA bping slect initiation codons?
- 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
How does the initiator tRNA bind to initiation codon?
- initiation codon lies in P site
- cells contain 2 distinct Met-tRNAs
- initiator tRNAs used to recognise initiation codon
- recognised internal AUG
How do initiator tRNAs vary in proks and euks?
- proks = tRNA met f, methionyl group formylated (addition of C=O bonded to H)
- euks = tRNA met, and isn’t modified
What cotranslationally modifies most euk proteins?
- N terminal acetyltransferase
How is translation initiated in E. Coli?
- 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
How is translation initiated in euks?
- 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
How is translation terminated?
- 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
What do many widely used antibiotics target?
- proks ribosome
How can toxins affect ribosome?
- 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
