BI236 Lecture Notes - Lecture 18: Aminoacyl-Trna, Non-Coding Rna, Peptidyl Transferase

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27 Jul 2016
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Protein synthesis and sorting: overview
For some genes, the RNA transcript is the final product
But for many other genes, the ultimate product is protein  mRNAs encode instructions for the
precise assembly of a.a into a polypeptide (translation)
Translation: the cast of characters
Ribosomes: carry out process of polypeptide synthesis
tRNA molecules align with a.a in the correct order
obring a.a to ribosome/mRNA complex
aminoactyl-tRNA synthetase: enzyme that attaches a.a to appropriate tRNA
mRNA: molecules encode a.a sequence info
protein factors: facilitate some of the steps of translation
The bacterial ribosome
2 dissociable subunits
Each subunit self-assembles from rRNA and proteins
oRibosome is 60% RNA, 40% protein
RNA does all the work and they come together only when binding the mRNA
Small subunit: 30S
Large subunit: 50S
Complete ribosome: 70S
Binding sites on a Ribosome
tRNA binding sites:
oA (aminoacyl site) : binds each newly arriving tRNA with its attached a.a
oP (peptidyl site): where tRNA carrying the growing polypeptide chain resides
oE (exit site): where tRNA leave ribosome after they have discharged their a.a
mRNA binding site
owhere subunits come together
obinds specific sequence at 5’ end
initiation factor binding sites
tRNAs bring a.a to ribosome
a tRNA molecule is an “adaptor” that binds both a specific a.a and the mRNA sequences that
specify an a.a
each tRNA is linked to it’s a.a by an ester bond (-O-)  high energy bond that will be used for
peptide bond formation
tRNAs are named for the a.a attached to them (e.g: tRNAAla for alanine)
tRNA structure
aminoacyl tRNA: tRNA with an attached a.a
nucleosides with modified bases:
The flexibility in codon-anticodon binding allows some unexpected base pairs to form:
oI = inosine (most common)
oml = methylinosine
oGm = methylguanosine
oT = ribothymidine
oD = dihydrouridine
o = pseudouridine Ѱ
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Wobble hypothesis, Francis Crick
The flexibility in codon-anticodon binding allows some unexpected base pairs to form
Explains how codons differing in 3rd position can code for same a.a
E.g: UUU, UUC = Phe (means 1 tRNA)
Degenerate nature of the code
Fewer tRNA than codons for same a.a (fewer are needed, instead of 6, 3)
Slight shift (“wobble”) in tRNA anticodon allows imperfect pairing with codon of mRNA (at 3rd base
only)
Inosine (I) is the wobbliest and often in the wobble position of the anticodon
oable to pair with U, C or A
does not cause insertions of incorrect a.a because diff codons recognized by a given tRNA always
code for the same a.a
Aminoacyl-tRNA synthesis
cells typically have 20 diff aminoacyl-tRNA synthetases to attach each a.a to the appropriate tRNA
oattachment avia ester bond using ATP hydrolysis
cells with nontraditional a.a have special tRNAs and aminoacyl-tRNA synthetases for these a.a
oselenocysteine; pyrrolysine
a.a activation by Aminoacyl-tRNA synthetase
synthetases recognize both anticodon and 3’ end of tRNA
ester bond linkage AA to tRNA is “high energy”
after addition, the synthetases proofread the final product to ensure the correct a.a was added
oAminoacyl-tRNA synthetase is crucial for accuracy
mRNA brings polypeptide coding information to the ribosome
Sequence of codons in mRNA directs order of a.a in polypeptide
mRNA is exported to the cytoplasm via binding to proteins that contain nucleus transport signals
(NES)
omRNA binding proteins that contain a.a sequences
An untranslated sequence at the 5’ end of the message precedes the start codon, the first to be
translated (usually AUG)
Comparison of bacterial and eukaryotic mRNA
5’ and 3’ untranslated regions (UTR)  vary in length and are essential for function
Start codon: where translation starts (N-terminus of polypeptide)
oFirst codon to be translated
Stop coon: where translation ends (C-terminus of polypeptide)
oCan be UAG, UAA, UGA
oLast codon to be translated
Included in untranslated regions of eukaryotic mRNA
o5’ cap (important in initiating translation in eukaryotes) & 3’ poly( A)tail
Eukaryotic mRNAs are mostly monocistronic
Most mRNA in eukaryotes are monocistronic
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oThey encode just 1 polypeptide
In bacteria and archaea, some are polycistronic
oEncodes several polypeptides usually with related functions
oOperons: Polysctronic transcription units
Cluster of genes that give rise to polycistronic mRNAs (single transcription units)
Overview of translation
Translation is an ordered, stepwise process
oInitiation, elongation, termination
mRNA is read 5’  3’
protein synthesis goes from N-terminus  C-terminus
Initiation of translation in bacteria
1. binding of IFs (IF1, IF2, IF3) & GTP to 30S subunit, with GTP attaching to IF2
2. binding of initiator tRNA (P site) and of mRNA to 30S subunit due to mRNAs ribosome binding
site(aka shine delgarno sequence; consists of a stretch of 3-9 purine ntds (AGGA) located slightly
upstream of start codon
oinitiator tRNA = fMet-tRNA (tRNAfMet)
in fMet, the amino group of methionine is blocked by the addition of a formyl
group and so cannot form a peptide bond with another a.a.
omRNA-binding site = part of 16S rRNA (specific sequence)
3. binding of 50S ribosomal subunit
orequires GTP hydrolysis
Eukaryotic initiation
the start codon in eukaryotes and archaea specifies methionine rather than N-formylmethionine
IFs are called eIFs (around 12 of them)
eIF2 (with GTP attached_ binds to initiator methioyl tRNAMet before the tRNA binds the small
ribosomal subunit (r40s)
oprokaryotes  IFs bind small ribosome subunit, then tRNA binds small ribosomal subunit
oeukaryotes IFs bind tRNA, then tRNA binds small ribosomal subunit
other IFs also bind small ribosomal subunit (eg: eIF1A, eIF3)
the resulting complex then binds to the 5’ end of the mRNA, recognizing the 5’ cap with the help
of eIF4F
oin some mRNA (eg: viral mRNA), the complex binds an internal ribosome entry sequence
(IRES) instead (upstream of start codon)
Afterwards, r40s (+ the initiator tRNA) scans along the RNA and begins translation at the 1st AUG
it finds
Nucleotides to either side of AUG are involved in recognition (eg: ACCAUGG = Kozak sequence)
After the pairing of the initiator tRNA-AUG, the r60s joins the complex, facilitated by GTP
hydrolysis
Elongation
1. Binding of 2nd aminoacyl-tRNA (+ EF-Tu-GTP) to A site
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