1
Q
Transfer RNA
A
-relatively small non-coding RNA, single stranded, approx 73-93 nucleotide residues long
2
Q
tRNA amino acid arm
A
- has nucleotide sequence 5’-CCA-3’ at 3’ term
- terminal A residue is where AA attach
- each tRNA carries specific AA, makes it aminoacylated (AA bound to it)
3
Q
tRNA anticodon arm
A
- at opposite end of tRNA to the AA arm
- 3 nucleotide sequence that base pairs complementary to the mRNA
4
Q
tRNA nomenclature
A
- Uncharged: tRNA^Leu
- Charged: Leu-tRNA^Leu
5
Q
“charging” of tRNA
A
- done by aminoacyl-tRNA synthetases
- the ribosome does NOT control if tRNA improperly charged
- different classes of synthetases have different points of contact - some have several, some don’t interact with the anticodon
- certain nucleotides on the tRNA code for specific synthetases to interact with the tRNA
6
Q
Wobble base pairing
A
- would expect 61 tRNA (64-3 STOP codons), is not the case, much fewer due to wobble
- still pyrimidine-purine, but orientation is slightly off (G-U)
- 1st and 2nd position of codon ALWAYS have Watson-Crick pairing
- 3rd codon position can have wobble
7
Q
Inosine
A
- wobble nucleotide, converted from adenosine through post-transcriptional modification of tRNA
- modification aided by ADAR (adenosine deaminase acting on RNA), hydration rxn and removes NH3
- CAN WOBBLE PAIR WITH A, C or U
8
Q
Crick’s rules of wobble hypothesis
A
- 1st 2 bases of mRNA codon ALWAYS Watson-Crick base pairs with corresponding tRNA anticodon
- A) when in 1st base of ANTICODON (in 5’-3’) is C or A, tRNA only recognizes one codon
- B) When the 1st base of the ANTICODON (5’-3’) is G or U, tRNA can recognize 2 different codons
- C) When the 1st base of the ANTICODON (5’-3’) is I, tRNA can recognize 2 different codons (A, U or C in the complementary position)
- codons that differ in 1st 2 bases require different tRNAs
- Minimum of 32 tRNAs required to translate all 61 codons
9
Q
Ribosome
A
- 60% rRNA, 40% r-protein, rRNA is functional component
- found free in cytoplasm or attached to endoplasmic reticulum
- Prok: 70S ribosome, Euk: 80S ribosome
10
Q
Ribosome subunits
A
- Small (30S/40S)
- contains 16S rRNA
- 21 proteins: RpS1-RpS21 - Large (50S/60S)
- 5S, 23S rRNA
- 36 proteins: RpL1-RpL36
11
Q
Svedberg Units
A
- S
- rate of sedimentation under a specific G force
12
Q
Ribosomal functional centers
A
- A Site: aminoacyl tRNA entry
- P Site: Peptidyl tRNA - growing peptide chain
- E Site: Exit site for uncharged tRNAs
- Decoding center: in small subunit, proofreading of codon-anticodon base pairing
- Peptidyl transferase center: in large subunit, transfer of polypeptide chain from peptidyl-tRNA in P site to aminoacyl tRNA in A site (no proofreading of charge of tRNA)
13
Q
Steps of translation (without termination)
A
- initiator tRNA charged with methionine
- translation initiates with assembly of mRNA and aminoacylated tRNA on the small subunit, large subunit then joins to form active ribosome
- polypeptide elongation occurs in successive cycles of aminoacyl-tRNA binding and peptide bond formation
14
Q
Initiation of translation
A
- most highly regulated stage of translation
1. alignment of mRNA on the small ribosomal subunit - IF-3 associates with the small subunit to prevent the premature assembly of the ribosome
2. Association of a charged initiator tRNA with the AUG start codon in the P site - tRNA moved to ribosome by IF-2
- IF-1 blocks A site to ensure correct alignment with AUG
3. Recruitment of large ribosomal subunit to form complete initiation complex - IFs dissociate from the complex (consumes GTP)
15
Q
tRNAs specific for Methionine
A
- tRNA^fMet is initiator tRNA, has a formyl group added to amino group of the aminoacyl tRNA
- N-formylmethionine
- blocks the Met on its N-terminus so it HAS to be the start, can’t add to carboxyl group
- tRNA^fMet interacts with IF-2
16
Q
Charging of tRNA^fMet
A
-2 step process:
-Met-tRNA synthetase charges both tRNA^Met and tRNA^fMet
-transformylase adds formyl group to Met, prevents charged tRNA from participating in elongation
Equivalent system in eukaryotes:
-tRNA^Met vs. tRNAi^Met
-tRNAi^Met interacts with eukaryotic equivalent of IF-2
17
Q
Shine-Dalgarno sequence
A
- in prokaryotes
- initiating AUG guided to correct position by Shine-dalgarno sequence
- 4-9 purines, 8-13 nt upstream of AUG
- has complementarity to 16S rRNA of small subunit
- also called ribosomal binding site (RBS)
18
Q
Kozak Sequences
A
- in eukaryotes
- consensus sequence surrounding the initiation site, aids in initiation
- NOT functional equivalent to RBS
- mRNA contacts the anticodon arm of Met-tRNAi^Met
- purine nucleotide 3 residues before start codon and G residue immediately following start codon thought to increase translation (through interaction with anticodon arm)
19
Q
Kozak vs. Shine-Dalgarno
A
- Shine-dalgarno associated with alignment
- Kozak interacts with anticodon arm of initiator tRNA
- both are conserved sequences in Prok/Euk but serve slightly different purposes
20
Q
Polycistrinsic mRNA
A
- multiple proteins from one mRNA
- ONLY in prokaryotes, Euk: 1mRNA = protein
- can have overlapping or non-overlapping genes
21
Q
Overlapping Genes
A
- no new Shine-Dalgarno for realignment
- open reading frames for the genes overlap, generally have overlapping start/stop codons (ie. AUGA)
- ribosomes terminating translation of upstream message can initiate downstream message by shifting reading frame
22
Q
Non-Overlapping genes
A
- the open reading frame for each gene is distinct
- genes have separate Shine-Dalgarno sequences
23
Q
GTP
A
- energy of translation
- guanosine triphosphate
24
Q
mRNA circularization
A
- exclusively in eukaryotes
1. mediates ribosome binding
2. promotes transcriptional efficiency
3. Ensures complete processing - each mRNA can be translated multiple times
- 1 mRNA bound by multiple ribosomes = polysome
BCHM 218 Part 2
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