27 Questions
Which of the following stop codons are recognized by RF-1 in termination of translation?
UAA and UAG
Which release factor recognizes all three stop codons (UGA, UAA, UAG)?
eRF-1
In coupled transcription and translation in bacteria, what is the direction of translation?
5' to 3'
Release factors act at which site of the ribosome during termination?
A site
Which of the following release factors is responsible for recognizing UGA and UAA stop codons?
RF-2
Which of the following is NOT a distinct step in translation?
Replication
How many nucleotides does a typical tRNA molecule contain?
73 to 93
Which molecule is NOT involved in the translation process?
DNA
What role do release factors (RFs) play during translation?
Terminate polypeptide synthesis
Which of the following is true about aminoacyl-tRNA synthetases?
They link amino acids to tRNA
Which step involves matching each amino acid with the correct tRNA?
Activation
Where does the initiation complex form in eukaryotic translation?
At the 5' terminus of the mRNA
What describes the optimal sequence for efficient translation initiation in eukaryotes?
Kozak's Rules
What is required to form the initiation complex in prokaryotes?
50S subunit of the ribosome
Which initiation factor binds to the 40S ribosomal subunit?
eIF1, 1A, 3 & 5
Which component binds the first amino acid in translation initiation in eukaryotes?
eIF2-GTP
What guides the mRNA to the 30S ribosomal subunit in prokaryotic translation initiation?
Shine-Dalgarno sequence
Which initiation factor mediates the binding of formylmethionine tRNA to the peptidyl site (P site)?
IF-2-GTP
What role does the eIF4F complex play during translation initiation?
It binds to the 5' CAP of the mRNA
How is the initiation complex completed in prokaryotic translation?
Attachment of large 50S subunit
Which antibiotic targets the elongation phase of bacterial protein synthesis?
Streptomycin
Which site on the ribosome does the initiator tRNA occupy during elongation?
P site
Which of the following antibiotics inhibit peptide-bond formation?
Linezolid
What is the function of the PolyA Binding Protein (PABP) during translation initiation?
It checks the ends of the mRNA
What happens to the A site during translocation in prokaryotic translation?
It becomes empty
What sequence is generally located 8 bp upstream of the AUG start codon?
Shine-Dalgarno sequence
Which of the following is NOT an initiation factor involved in prokaryotic translation initiation?
EF-Tu
Study Notes
Termination
- STOP codons: UGA, UAA, and UAG
- No tRNAs for these codons
- Recognized by release factors (RF)
- RF-1 for UAA and UAG
- RF-2 for UGA and UAA
- eRF-1 recognizes all 3 codons
- RF acts at A site and needs an occupied P site
- RF causes the chain to be released from the P site tRNA
- RF then escapes from the complex
Coupled Transcription and Translation in Bacteria
- Transcription occurs from 3' to 5' end
- mRNA is translated by a ribosome from 5' to 3' end
- Ribosome is composed of two tRNAs, each with a +NH3 attached
Translation - The Process
The Macromolecules of Translation
- Polypeptides (>50) and RNA molecules (3-5) make up the ribosome
- Amino acid activating enzymes (20)
- tRNA molecules (32-60)
- Soluble proteins involved in polypeptide chain initiation, elongation, and termination
- ATP and Mg2+
Distinct Steps in Translation
- Initiation
- Elongation
- Termination
tRNA
- 73 to 93 nucleotides long
Stage 1: Activation of the tRNA
- Catalyzed by aminoacyl-tRNA synthetases
- Each enzyme links a specific amino acid to the matching tRNA
- 20 different aminoacyl-tRNA synthetases (1 for each amino acid)
- Some cells contain less than 20 synthetases, in which case one amino acid is converted to another after charging the tRNA
- Matching each amino acid with the correct tRNA is the "second genetic code"
- Codon specificity resides in the tRNA, not the attached amino acid
Stage 2: Initiation in Prokaryotes
Ingredients
- 30S subunit of the ribosome
- Initiator tRNA (tRNAMet)
- mRNA
- Initiation Factors IF-1, IF-2, and IF-3
- One molecule of GTP
- 50S subunit of the ribosome
Formation of the Initiation Complex
- mRNA is guided to the 30S ribosomal subunit by the Shine-Dalgarno sequence
- Shine-Dalgarno sequence is a ribosomal binding site in the mRNA
- Generally located 8 bp upstream of the AUG start codon
tRNA Binding Sites on the Ribosome
- E or exit tRNA
- P or peptidyl binding site
- A or aminoacyl binding site
Formation of the Initiation Complex
- IF-1, IF-2, and IF-3 are initiation factors
- P site - peptidyl site
- A site - aminoacyl site
- E site - exit site
Translocation
- Elongation needs another elongation factor for translocation
- All amino acids move along one space, resulting in an empty A site
- When the A site is filled, the E site is emptied
- The 3rd EF recycles the first elongation factor
- This continues until a STOP codon is reached
Elongation
- All ribosomes have 3 sites: A, P, and E
- Initiator tRNA is in P site
- Next amino acid is escorted to A site
- The small subunit and base pairing "check" amino acid is correct
- Correct amino acid causes conformational change and hydrolysis of bound GTP
- EF's released and peptide bond formed
Translation Initiation in Eukaryotes
- The amino group of the methionine on the initiator tRNA is not formylated (i.e., Met, not fMet)
- The initiation complex forms at the 5' terminus of the mRNA
- No zone of Shine-Dalgarno--AUG start sequences
- The initiation complex scans the mRNA for an AUG initiation codon
- Translation usually begins at the 1st AUG found
- Kozak's Rules describe the optimal sequence for efficient translation initiation in eukaryotes
Stage 2: Initiation in Eukaryotes
- Prepare the Ribosome
- 40S subunit binds initiation factors (eIF1, 1A, 3, and 5)
- Prepare the 1st amino acid
- tRNA-Met binds to eIF2-GTP
- Prepare the RNA
- eIF4 family + PolyA Binding Protein (PABP) check ends of mRNA
- All merge, ATP -> ADP, all factors released
- 60S subunit binds
Antibiotics Target Ribosome Sites During Bacterial Protein Synthesis
- Initiation: Edeine (Ede), kasugamycin (Ksg), pactamycin (Pct), thermorubin (Thb) (30S subunit)
- Initiation: orthosomycins avilamycin (Avn), evernimicin (Evn), thiostrepton (Ths) (50S subunit)
- Elongation (by EF-Tu): inhibited by streptomycin (Stp), tetracyclines (Tet), glycylcyclines (tigecycline (Tig))
- Peptide-bond formation: blasticidin S (Bls), chloramphenicol (Cam), lincosamides (clindamycin (Cln)), oxazolidinones (linezolid (Lnz)), pleuromutilins (Plu), puromycin (Pmn), streptogramin A (S) and sparsomycin (Spr)
- tRNAs translocation (by EF-G): tuberactinomycins capreomycin (Cap), viomycin (Vio), hygromycin B (HygB), neomycin (Neo), paromomycin (Par), fusidic acid (Fus), spectinomycin (Spt), Ths
- Chain elongation: macrolides (erythromycin (Ery)), streptogramin B (S), ketolides (telithromycin (Tel))
- Termination: peptidyl-transferase inhibitors Bls, Cam, Pmn, Spr
- Recycling: translocation inhibitors, especially Fus
This quiz covers the termination stage of protein synthesis, including STOP codons, release factors, and the process of chain release. It also touches on coupled transcription and translation in bacteria.
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