Lecture #7 Flashcards
What is Translation?
Genetic information is converted into protein information.
- Generation of peptide strand from a nucleotide sequence.
- Template: mRNA
Properties of Translation
Not a 1:1 association:
- 4 nucleotides vs. 20 amino acids
Rule: a codon of nucleotides is needed for each amino acid.
- A group of three consecutive nucleotides in an mRNA strand.
- 64 possible combinations
Some amino acids use more than one codon.
Redundancy
Properties of Translation Continued
A given mRNA sequence can be translated using one of three different “reading frames”.
- Why does it matter which reading frame is used?
Rule: reading frame begins at the AUG site.
- Start codon: AUG
Translation requires the presence of “adaptor” proteins.
- tRNAs
tRNAs act as adaptors during protein synthesis
tRNAs
- transfer RNAs
- Made during transcription
- Small RNA molecules
Form double helix regions
- Base pairing
- provides additional folding
- anti-codon loop
tRNAs act as adaptors during protein synthesis continued
tRNAs
- One tRNA can base pair with more than one codon.
- Only need ~ 31 tRNAs to “read” the possible codons.
- “Wobble Position”
Position 3( 5’ position)
- “Wobble Position”
Aminoacyl-tRNA synthetases attach amino acids to tRNAs
aminoacyl-tRNA synthetase
- Attaches amino acid to the 3` phosphate group on the tRNA.
- Different synthetase is used for each amino acid.
- Recognition based on nucleotides in the anticodon and in the amino-acid-accepting arm.
Ribosomes and Translation Part I
Ribosomes facilitate the rapid and accurate addition of amino acids to the growing polypeptide chain.
Ribosomes and Translation Part II
Ribosome
- The site of protein translation
- Cytoplasm of the cell or the ER
- Millions present within the cell
4.2 MDa
2/3 RNA + 1/3 Protein
- 50 proteins
- 4 rRNA molecules
Large Subunit
Small Subunit
Ribosomes and Translation Part III
Ribosome
Core - rRNA
- Determines ribosome structure.
Proteins located on the surface
- Fills in the gaps
- Stabilize the rRNA core
23S rRNA catalyzes the addition of an amino acid to the growing strand – peptidyl transferase
- Ribozyme
Ribozyme: RNA molecules capable of catalytic activity.
The ribosome and polypeptide chain growth
The ribosome contains…
One binding site for the mRNA
- small subunit
Three binding sites for three tRNAs
A-site – aminoacyl tRNA
P-site – peptidyl tRNA
E-site – Exit site
The Four Steps of Translation Step One
Step One aminoacyl tRNA binds in the A-site tRNA is “charged” - tRNA is "charged" - Anticodon sequence is complimentary to mRNA codon sequence.
“A” and “P” sites are close together
- prevents base pair skipping
tRNA in E-site is ejected
The Four Steps of Translation Step Two
Step Two
- Amino acid chain is uncoupled from the P-site tRNA
- Peptide bond formed between the peptide chain and the amino acid attached to the tRNA in the A-site
- Catalyzed by the rRNA within the large subunit
The Four Steps of Translation Step Three
Step Three
Large subunit translocates 5→3
tRNA in the P-site moves to E-site of the large subunit
tRNA in the A-site moves to the P-site of the large subunit
The Four Steps of Translation Step Four
Step Four
Small subunit translocates
- Mediated by base pairing in E-site and P-site.
Empty A-site is available for next charged t-RNA
**Remember** Translation - 5'- 3' Peptide strand generation - N-terminis--> C-terminis
How do we get started? Part I
Translation initiated by an initiator tRNA
Carries amino acid Methionine
- Different from others
Charged initiator tRNA + translation initiator factors bind to the small ribosomal subunit
- P site
Binds to the 5` end of the mRNA
- How long does it know which end?
How do we get started? Part II
Moves along mRNA in 5→3
Stops at the AUG codon.
- What’s the anticodon sequence?
Initiator factors dissociate.
Large ribosomal subunit binds to the small ribosomal subunit.
How do we stop?
Stop codons are not recognized by any tRNAs.
- UAA, UAG, UGA
Release factors bind to the A-site in the absence of tRNAs.
Peptidyl transferase catalyzes the addition of H2O instead of an amino acid.
Carboxyl end of the amino acid chain is freed.
- Peptide chain is released into the cytoplasm.
mRNA is released and ribosome dissociates.
Are there any differences in bacteria?
Bacteria do not have 5` caps on their mRNA
Bacteria contain ribosome binding sites (rbs) in their mRNA
- Shine-Dalgarno sequence (AGGAGG)
- 6-7 nucleotides long
- Upstream of the start codon.
- Don’t rely on tRNAs to bind to mRNA.
mRNAs are polycistronic
- multiple genes on one mRNA strand.
Many proteins are made quickly using polyribosomes
Assembly of many ribosomes on a single mRNA strand.
- can be as close as 80 nucletotides a part.
A second ribosome sits down as soon as the first ribosome exits the start codon.
Generates multiple peptide strands at different points during synthesis.
What happens next?
Some peptide strands fold spontaneously.
- Interactions between the amino acid side chains.
Some peptide strands require molecular chaperones to fold properly.
- Larger proteins w/ multiple “domains”.
Regulation of protein levels within the cell Part I
Protein degradation pathways
- Lifetimes – differ for each protein
- Misfolded or damaged proteins
The lysosome contains proteases
- Enzymes which hydrolyze the peptide bond between amino acids.
The proteasome degrades proteins within the cytosol
- Central cylinder capped by two “stoppers”.
- ATP is used to move the proteins into the cylinder for degradation.
Regulation of protein levels within the cell Part II
Proteins are “tagged” for degradation by a specific post-translational modifications
- Ubiquitination
Ubiquitin protein is attached to specific lysines in the protein sequence
Proteins can be mono- or poly-ubiquitinated
- Poly-ubiquitination signals degradation.
Protein expression regulation
Many cellular processes can be used to regulate protein levels within the cell.
- Before and after peptide strand generation.
