Gene Expression (3) and Protein Synthesis (translation) and Sorting Flashcards
What are the 5 major components involved in translation? What do each do in translation?
- Ribosomes: carry out the process of polypeptide synthesis
- tRNA: align the amino acids in the correct order
- Aminoacyl-tRNA synthetases - attach amino acids to their appropriate tRNA molecules
- mRNA molecules - encode the amino acid sequence information
- Protein factors - facilitate some of the steps of translation
What can be noted about bacterial ribosomes?
- Made of two dissociable subunits; large subunit and small subunit
- Each subunit self-assembles from rRNA and proteins
- they come together only when binding mRNA
How many binding sites are on a ribosome?
-Four sites of binding
-Three tRNA binding sites one mRNA
-A (aminoacyl) site – binds each newly arriving tRNA with its attached amino acid
-P (peptidyl) site – where the tRNA carrying the growing polypeptide chain resides
-E (exit) site – from which tRNAs leave the ribosomes after they have discharged their amino acids
-mRNA binding site is where subunits come together
binds specific sequence at 5’ end
There are also initiation factor binding sites
What does the tRNA do? Why are they important?
- tRNA brings amino acids to the ribosome
- A tRNA molecule - an adaptor that binds both a specific amino acid and the mRNA sequences that specify the amino acid
- Each tRNA is linked to its amino acid by - an ester bond (–O-)
- named for the amino acids attached to them, e.g., tRNAAla for alanine
- Once the amino acid is attached, the tRNA is called amino acyl tRNA (eg. alanyl tRNAAla)
- The tRNA is said be – “charged” form when amino acid attached or “activated”
How does translation start?
- mRNA is exported to the cytoplasm via binding to mRNA-binding protein that contains amino acid sequence nuclear export signals (NES) for transport through the nuclear pores (not required in prokaryotes, as no nucleus)
- An untranslated sequence at the 5’ end of the message precedes the start codon, the first to be translated (usually AUG)
What’s the difference in Bacterial and Eukaryotic Messenger RNA?
Eukaryotes have a cap and poly A tail
Bacteria do not
What are the three steps of Initiation of translation in bacteria?
Step 1 – Three initiation factors (IF1, IF2 and IF3) and GTP bind to the small ribosomal subunit
Step 2 – The initiator aminoacyl tRNA and mRNA are attached and bind to the small ribosomal unit
initiator tRNA is tRNA that carries an N formylmethionine→ tRNAfMet (only bacteria have this, not eukaryotes)
Step 3 – The large subunit joins the complex resulting 70S initiation complex and GTP is hydrolysed
Translation Initiation: what is the difference between bacteria and eukaryotes?
- The start codon in eukaryotes and archaea requires methionine
- Start codon requires N-formylmethionine in bacteria
- Eukaryotes use a different set of initiation factors known as eIFs; there are about a dozen of these as eIF1, eIF2
- Eu. and Pro. have different pathway for assembling the initiation complex
- Euka use initiator tRNAMet, that carries methionine that does not become formylated
How does eukaryotic initiation begin?
- eIF2 (with GTP attached) binds to the initiator methionyl tRNAMet, then binds to the small ribosomal subunit
- Other initiation factors also bind to the small subunit (e.g., eIF1A)
- The resulting complex then binds to the 5; end of the mRNA, recognizing the 5’ cap with the assistance of a cap-binding initiation factor, eIF4F
How does eukaryotic initiation begin?
- After binding to mRNA, the small ribosomal subunit (including the initiator tRNA) begins translation at the first AUG triplet it encounters
- The nucleotides on either side of the start codon are involved in the recognition; e.g., a common start sequence is ACCAUGG, (Kozak sequence)
- After the initiator tRNAMet is base-paired with the start codon, the large subunit binds to the complex and initiation starts
How does chain elongation occur?
- 3-step cycle
- Aminoacyl tRNA binding – ribosome bring a new amino acid
- Peptide bond formation – links this amino acid to the growing polypeptide chain
- Translocation – mRNA is shifted over three nucleotides using translocation to bring the next codon into position for translation
How does termination occur?
- stop codon (UAA, UAG or UGA) arrives at A site; recognized by ‘Release factors’ proteins coupled to GTP
- Hydrolysis releases the polypeptide (release is due to a hydrolytic cleavage reaction)
- Ribosomes dissociate into its subunits; tRNAs and mRNAs are released
What posttranslational processing occurs?
- the polypeptide chain is chemically modified before can perform their normal functions
- In bacteria : N-formyl group at the N terminus (free amine group located at the beginning of a polypeptide chain) of chain and Met attached to it, are removed
- In eukaryotes : Met at the N-terminus is removed
- Sometimes, whole blocks of amino acids are removed
- Some enzymes synthesize as inactive precursors, and must be activated by removal of specific sequences at one end or the other
What posttranslational processing occurs? cont.
- Internal stretches of amino acids - removed to produce an active protein
- Chemical modifications of individual amino acid groups (methylation, phosphorylation or acetylation)
- Polypeptides may undergo glycosylation or binding to prosthetic groups
- individual polypeptide chains bind together the multisubunit proteins or multi-protein complexes
How does protein splicing occur? What are the two types?
- Splicing can intramolecular or intermolecular
- Inteins (certain amino acid sequences) - removed from a one or more polypeptides
- Resulting segments are spliced together to create a continuous polypeptide chain
