Midterm #2: Translation Flashcards
1
Q
Translation Overview
A
- Proteins are synthesized by ribonucleprotein complexes called ribosomes from mRNA
- Genetic code: relationship between the sequence of bases in DNA, it’s mRNA transcript and the sequence of amino acids in a protein
- U replaces T in RNA
- A codon is the three nucleotide bases in an mRNA transcript
2
Q
The Genetic Code
A
- Each codon codes for a single, specific amino acid
- Many amino acids are coded for by more than a single codon
- The genetic code is degenerate
- Three codons code for the termination of protein synthesis and are called stop codons
3
Q
Point Mutations
A
- Changing a single nucleotide in the sequence results in a point mutation
- Silent mutation: the same amino acid specified
- Missense mutation: a different amino acid is specified
- Nonsense mutation: turns an amino acid codon into a stop codon
- Read-though mutation: turns a stop codon into an amino acid codon
4
Q
Errors in the code: Frameshift mutation
A
- Addition or deletion of a single base in the mRNA transcript
- Changes the identities of all subsequent residues in the protein
5
Q
Chemistry of Amide Bond Formation: A condensation reaction
A
- Energetically unfavorable reaction, delta G is greater than 0
- Energy barrier overcome by activating the amino acid
- The activated intermediate is an aminoacyl-tRNA
6
Q
Amino Acid Activation
A
- AMP and PPi as by-products
- Activated for polymerization into polypeptide chain by enzymes called aminoacyl-tRNA synthetases
- Attach a specific tRNA molecule to the carboxylic functional group
- Note that 2 high-energy bonds are hydrolyzed to activate each amino acid
7
Q
Ribosomes: The Engines of Translation
A
- Very large (~2700 kDa)
- Ribonucleoprotein proteins of approximately 200 angstroms
- Large 50S subunit and small 30S subunit
- Are approximately 20,000 ribosomes in an E. coli
8
Q
Ribosomes have 3 functionally distinct tRNA-binding sites
A
- Aminoacyl (A) site: accommodates the incoming aminoacyl-tRNA
- Peptidyl (P) site accommodates the peptidyl-tRNA (nascent polypeptide)
- Exit (E) site accommodates a deacylated tRNA that is about to exit the ribosome
9
Q
Initiation of Translation in Prokaryotes: Shine-Delgarno Sequence and anti-codon
A
-
Shine Delgarno Sequence:
- purine rich
- Defines the initiation codon in prokaryotic mRNA
- protein synthesis initiates at a downstream AUG codon
- Recognized by the 16s RNA (part of the 30s which is part of the 70s RNA)
- The anti-codon of tRNAfMet recognizes an AUG codon
- Protein synthesis always starts at and AUG (met) codon, but not all AUG initiate translation
- during elongation, AUG is recognized by tRNAMet
10
Q
Initiation Complex in Prokaryotes
A
- Initiation complex is composed of the small ribosomal subunit, mRNA, and fMet-tRNAfMet
- Ribsomes assembly requires proteins called initiation factors
- The Shine-Delgarno sequence in the mRNA base pairs with the 16S rRNA in the small (30s) ribosome subunit
- Note that the only thing required to initiate translation of prokaryotic mRNA is the Shine-Delgarno sequence. If a single mRNA strand has 3 S-D sequenuences, 3 protiens will be translated.
- **polycistronic genes **
11
Q
Initiation of Tranlation in Prokaryotes
A
- AUG codon placed in the P site where fMet-tRNAfMet enters
- represents the “nascent” peptide chain
- The 50s subunit binds the mRNA*fMet-tRNAfMet*30s subunit complex
- poised to bind a second aminoacyl-tRNA in the A site
- Note that GTP hydrolysis by IF2 is required to assemble the functional ribosome
12
Q
Polypeptide Elongation
A
- activated tRNA is delivered to the A site by elongation factor EF-Tu
- if the tRNA anticodon is NOT properly paired, EF-Tu•GTP and the aminoacyl-tRNA dissociate from the ribosome.
- If the tRNA anticodon is properly paired with the A-site codon, EF-Tu hydrolyzes it bound GTP and dissociates from the ribosome.
- powered by GTP hydrolysis
- fidelity of bond formation is 10-4 errors/codon
13
Q
Peptidyl Transferase
A
-
peptidyl transferase activity of 50s ribosomal subunit catalyzes a trans-peptidation reaction
- catalyzed by the 23s rRNA- a ribozyme (ie: an RNA with catalytic activity)
- free amino group of aminoacyl-tRNA at the A-site attacks the activated carbonyl of the amino acid at the P site
- Lengthens nascent peptide by one amino acid
- Note that the nascent peptide chain grows in the N→C direction
14
Q
Translocation
A
- The ribosome slides one codon to the right (towards the 3’ end) on the mRNA
- powered by GTP hydrolysis
- places peptidyl-tRNA in the P-site and the uncharged tRNA in the E site (dissociates)
- the ribosome•mRNA•peptidyl-tRNA complex is now ready for EF-Tu to deliver the next aminoacyl-tRNA to the A-site and subsequent peptide bond formation
15
Q
Polyribosome
A
- Because mRNA is syntheized in the 5’→3’ direction, and because ribosomes read mRNA in the 5’→3’ direction, prokaryotic ribosomes can commence translation as soon as mRNA emerges from RNA polymerase
- A second ribosome can bind to an mRNA as soon as the first one has cleared the initiation site
- multiple ribosomes to a single mRNA transcript gives rise to a polyribosome
16
Q
Termintation of Translation
A
- There are no tRNA molecules that recognize the codons UAA, UGA, or UAG.
- These are STOP codons that are recognized by release factors
- bind to A site and trigger hydrolysis of the protein from the tRNA, which leads to dissociation of the ribosome
- Dissassembly requires one GTP
23
Q
Aminoglycosides bind to the A site
A
- Note the 2 bases flipped out of the helix
- indicates that the correct tRNA is in the A site
- Aminoglycoside lock the A site into this conformation
- preventing recognition of a mismatch and impair proofreading
