Main Ideas - The genetic codes specifies correspondence between codons and amino acids: it is universal, degenerate, adnd unambigous !- Translation requires coordination between the mRNA, tRNAs and the ribosome (rRNA)!- tRNAs connect the mRNA with the protein sequence !- aminoacyl tRNA synthetases attach the correct aa to the correct tRNA!- the ribosome is a complex catalytic machine comprised of sever rRNAs and >50 proteins arranged in 2 subunits, a large and a small!- the small ribosomal subunit binds to the 5' end of the mRNA and scans it to find the first AUG!- the ribosome holds 2 tRNAs at a time, one in the P (peptidyl site), and one in the A site (aminoacyl)!- the ribosome moves along the mRNA 3 nucleotides at a time. It stops when it encounters a stop codon, which is bound by a protein release factor!- many antibiotics work by selectively targeting bacterial translation!Learning Objectives 1) Understand the main features of the genetic code 2) Describe structure and function of transfer RNAs 3) Describe major components and steps of protein synthesis 4) Explain how protein synthesis is regulated 5) Discuss diffs betwen euk and prok translation and the basis for the use of many antibiotics 1) Understand the main features of the genetic code - mRNA is read in consecutive groups of 3= codons!- 4 diff. nucleos= 4x4x4= 64 diff. codons...But we only have 20 aa commonly found in proteins!- start: AUG (methionine)!- stop: UAA, UAG, UGA!- the genetic code is pretty much universal (sans for some rando species + mitochrondria)!- it is degenerate (most aa have more than 1 codon) and unambigous (each codon codes for only 1 aa)!- it is non-overlapping and w/o punctuation!- the start codon determines the reading frame!- the coding sequence can be changed by:!- point mutations (silent, missense, non-sense= change aa to stop codon)!- deletions or insertions (if can't dividie by 3--> frameshift mutation) 2) Describe structure and function of transfer RNAs - 3' end has the aa, anticodon in middle binds to complementary codon on mRNA!- are transcribed by RNA poly III-->precursor tRNA!-pre-RNAs are trimed and have introns spliced out by exonucleases (cut and paste)!- tRNAs are heavily modified before they leave the nucleus!- U--> dihydrouridine, pseudoU!- methyl groups added to purines!- ribose methylated at 2-OH!- Some aa's have more than 1 tRNA and some tRNAs allow for a mismatch at the 3rd nucleotide in the codon= wobble base-->thus many cells have fewer that 61 tRNA because w/the wobble base, one tRNA can recog. several diff. codons that specify a given aa!- Each aa is coupled to the right set of tRNAs (put on at the 3' end) by aminoacyl-tRNA synthetase!- each aa has its own aa-tRNA syn.!- this coupling reqs ATP hydrolysis!- tRNA synthetase can proofread!!- 1 mistake/40,000 couplings!- correct aa has the highest affinity for the active site on the aatRNA syn.!- also, when the coupling is happening, the tRNA tries to move the aa to another site on the aa-tRNA syn.-->the editing site, but only incorrect aa's fit into the editing site-->they will then be hydrolyzed from the tRNA--> try again tRNA! 3) Describe major components and steps of protein synthesis - 3 types of RNAs for translation!- mRNA (carries the genetic info), rRNA (catalyst of protein syn. in the ribs), and tRNA (brings that aa's to the ribs)!- the template for translation if the mature mRNA!- contains only exons (coding region), 5' cap, 5' UTR (untranslated region), 3'UTR, and poly-A tail!- has translation start and stop signals!- has to have the intact/correct reading frame to be correctly translated into the right aa sequence - the ribosome! many diffs between prok and euk aka they can be target for antibiotics!! Steps of Translation - Peptide chain initiation!- Peptide chain elongation!- Peptide chain termination! Initiation of protein synthesis - reqs. eukaryotic initiation factors (eIFs)!- mRNA has to be ready to go: 5' cap, poly A !tail covered in poly-A binding proteins!- translation begins at an AUG (Methionine) in the context of the Kozak consensus sequence!- when AUG is found, the eiFS leave and let the large rib subunit bind !- when the ribosome is assembled, the next aminoacyl-tRNA enters and bp's with the 2nd codon via its anticodon seq.!- the 1st peptide between Met and the 2nd aa forms and we shift gear into elongation!!!!!Peptide Chain Elongation - 1. an aminoacyl tRNA binds the vacant A site (via its anticodon)!- 2. a new peptide bond is catalyzed between the two tRNAs by peptidy transferase (= a part of the rRNA, it's not a protein, = 28S rRNA in the 60S large subunit)!- 3. the large sub moves over and shifts that tRNAs into the E and P sites!- 4. the small subunit with its bound mRNA shifts 3 nucleotides over!- the ribosome is ready for its next aminoacyl tRNA Peptide Chain Termination - end of translation is signaled by a stop codon (UAG, UAA, UGA) on mRNA!- stop codons are recog. by a tRNA, they are recog. by a release factor that binds to the A site!- release factors look just like tRNAs! sneaky!- release factor forces peptidyl transferase to add H20 to carboxyl end of the peptide chain!- the chain is releasd, and then the mRNA is released-->thus is can be used again for synthesis!- Internal ribosome entry sites (IRES)!- 90% of euk mRNAs are translated the reg. !way, some euk mRNAs start translation !using an IRES!- Viruses use IRES to block the 5'CAP !dependent translation of cellular mRNAs!!- initiate translation from an internal mimicry start site! - Proteins are synthesized on poly ribosomes - next rib binds as soon as the AUG is available!- the 5' and 3' end of the mRNA stay connected-->easy reassembly of the ribs!- syn: 20 sec- a few minutes! 4) Explain how protein synthesis is regulated - Translation efficiency is regulated by:!- accuracy of the mRNA molecule!- the half-life of the mRNA molecule!- the probabilty of initiating translation (aka how good is the consensus seq)!- rate of protein synthesis (ex. eIF2 activity is stimulated by dephosphorylation)!- Nonsense-mediated mRNA decay - most powerful surveillance system for abnormally spliced mRNA-->it checks on mRNA during the 1st round of translation!- introns have stop codons in all the possible reading frames, so an intron in the mature mRNA(incorrectly there) will contain an in frame stop codons, but exon junction complexes (EJCs) will be bound downstream of that stop -->cell knows that is wrong--> mRNA degradation! 5)
View Full Document
Unlocking...