MBB222 - Lecture 32: Protein synthesis (pt.2)

what are the steps in the formation of the prokaryotic translation initiation complex (3 steps)

• GTP, IF1, IF2, IF3 → bind to the 30S ribosomal subunit

• mRNA and fMet-tRNA_i ^met binds to the complex with the Shine-Dalgarno sequence of mRNA aligning with complementary sequences in the 16S rRNA in the 30S ribosomal subunit → IF3 is released

• hydrolysis of GTP facilitates binding of the 50S ribosomal subunit - release of IF1 and IF2 forms the function 70S ribosomal initiation complex

list the 6 steps for translational elongation

what does the 6 step elongation cycle require

requires the hydrolysis of 2 GTP

• one used to promote binding of the AA-tRNA^AA to the A site

• one to facilitate translocation of the ribosome in the 3’ direction

the peptidyl transferase reaction is catalyzed by what

the ribozyme activity of the 23S rRNA in the large ribosomal subunit

what is the peptidyl transferase reaction

• nucleophilic amino group of amino acid bound to the 3’ terminus of A-site tRNA → attacks the carbonyl carbon in the ester bond

• rearrangement of the tetrahedral intermediate results in transfer of the formerly P-site amino acid ( or polypeptide) to the amino acid on the A site that is bound to tRNA

which direction does ribosome “track” along the mRNA

5’ → 3’ direction - synthesizing proteins in the N→ C terminal direction

what are the binding sites for tRNA

A- / P- / E-sites → span in both large and small ribosomal subunit

how do new polypeptides exit from the ribosome

newly-synthesized polypeptide exits through a hydrophobic tunnel in the 50S subunit at the site of peptide bond formation

what happens during the termination of translation

release factors (RF) bind to the stop codon and lead to release of the polypeptide chain

can occur 2 ways:

• binding of the release factor protein (RF2) to the stop codon in mRNA leads to GTP hydrolysis → releases all bound translational components

• molecular structure of the 70S bacterial ribosome with tRNA molecules in the E and P sites + RF2 in the A site + short mRNA substrate that has the stop codon

what are the differences between prokaryotic and eukaryotic translation initiation complexes

eukaryotic lack a specific ribosome binding site in the mRNA + has a greater number of eukaryotic initiation factors (eIFs) required

what are examples of 4 antibiotics that inhibit bacterial protein synthesis at specific steps

chloramphenicol → binds to the 50S subunit and prevent peptide bond formation

Erythromycin → binds to the 50S subunit and prevents translocation

Tetracyclines → prevents tRNA binding to the A site in the ribosome

Streptomycin → alters the structure of the 30S subunit - causes errors in translation

what are polyribosomes/polysomes

group of multiple ribosomes attached to a single mRNA molecule

where are proteins made on

polyribosomes/polysomes

how can multiple synthesis occur on a single mRNA

through polysome formation - multiple ribosomes translate the same strand simultaneously

what is co translational folding of proteins

the process where a protein starts to fold into its active 3D shape while it is still being made by a ribosome

why do some proteins need to be post translationally modified

in order to adopt their biologically active form

what is proteasome - what does it do

an ATP dependent protease complex that rapidly destroys misfolded proteins

• proteins with exposed hydrophobic regions → marked for destruction by covalent binding of multiple copies of a protein (ubiquitin)

• protein marker allows them to be recognized by proteasome

• misfolded proteins are bound by proteosome → unfolded → degraded into short peptides