BY450 Gene Expression & Regulation - Translation (PDF)

BY450 Nature and function of the Gene 2: Gene expression and regulation Dr Nigel Brissett Session 3 mRNA to protein: translation (ctd.) Components of translational process amino acyl tRNA ribosome large unit...

BY450 Nature and function of the Gene 2: Gene expression and regulation Dr Nigel Brissett Session 3 mRNA to protein: translation (ctd.) Components of translational process amino acyl tRNA ribosome large unit mRNA ribosome small unit Translation- covered so far: tRNA structure and synthesis Ribosome structure- eukaryotic, prokaryotic rRNA, ribosomal proteins  tRNA structure Figure 7-28 Essential Cell Note: 70-80 nucleotides long, complementary base pairing (loops), Biology (© Garland Science modified bases (eg pseudouridine, dihydrouridine) 2010) Linkage of amino acid to tRNA 1. amino acid eg alanine coupled to AMP catalysed by family of aminoacyl tRNA synthetases (36 tRNA distinct species/cell) 2. aminoacyl AMP combines with tRNA to form aminoacyl tRNA Translation- covered so far: tRNA structure and synthesis Ribosome structure- eukaryotic, prokaryotic rRNA, ribosomal proteins Ribosomal RNA structure/sequence E.coli 16S rRNA Structural scaffold for proteins Catalytic activity (eg peptidyl transferase of large su) Ribosomes consist of RNA and proteins green 50S protein (light green RNA) blue 30S protein (light blue RNA) Overview of translation- 3 stages Translation initiation- prokaryotes, sequence signals, RNA-RNA interactions ‘Shine-Delgarno’ mRNA sequence (precedes AUG codon): aligns with 30S ribosomal 16S rRNA Translation initiation- eukaryotes, sequence signals, RNA-RNA interactions eukaryotic mRNAs bind to 40S ribosomal subunit via 5’ 7-methylguanosine cap with help of translation factors  Translation is mediated by a complex set of translation factors Role Prokaryotes Eukaryotes Initiation IF1 eIF1, eIF1A, eIF2, eIF2B, IF2 eIF3, eIF4A, eIF4B, IF3 eIF4E, eIF4G, eIF4H, eIF5, eIF5B Elongation EF-Tu, eEF1a EF-Ts eEF1bg EF-G eEF2 Termination RF-1, RF-2, RF-3 eRF1, eRF3 Cooper (2016), Table 9.1 Translation initiation in prokaryotes 1 Initiation factors (IF1, IF2, IF3) bind to 30S subunit together with mRNA and fMet-tRNA (amino acyl tRNA) Translation initiation in prokaryotes 2 50S subunit joins complex and IF1 and IF3 released; IF2 released 70S mRNA-ribosome complex ready for translation Overall requirements: mRNA, amino-acyl tRNA (fmet), ribosome, GTP, 3 protein factors  Translation initiation in eukaryotes requires mRNA, amino-acyl tRNA (met), ribosome, GTP, 12 protein factors 1. translocation along mRNA ……AUG identified  Translation initiation in eukaryotes requires mRNA, amino-acyl tRNA (met), ribosome, GTP, 12 protein factors 2. release of translation initiation factors 3. 60S subunit joins 40S/tRNA complex 4. 80S ribosome- met tRNA initiation complex completed  Elongation- takes place in 4 steps Similar process prokaryotes and eukaryotes- different elongation factors ribosome has 3 tRNA binding P (peptidyl) sites A (aminoacyl) E (exit)  Elongation Step 1 at translation initiation met tRNA present at P site  Elongation 2 Step 2 amino acyl tRNA for next codon enters A site facilitated by eEF1a* * EF-Tu in prokaryotes  Elongation 3 Step 3 Carboxyl end of amino acid is uncoupled from the tRNA at P-site and peptide bonded to the free amino group of amino acid linked to tRNA at A-site  Elongation 4 Step 4 Shift in ribosome moves the two tRNAs one place to the E- and P-sites of the large subunit. Requires eEF2* Uncoupled tRNA released From E site Process repeats to form growing polypeptide chain * EF-G in prokaryotes Termination Stop codon reached- no complementary tRNA.  Termination Stop codon reached- no complementary tRNA. Release factors bind to the A-site (eRF1/eRF3*) *RF1/RF3 RF2/RF3 in prokaryotes Termination Polypeptide chain is released by hydrolysis of bond with tRNA in P site Termination tRNAs released and ribosome dissociates  Topic overview Transcription mRNA processing Translation Control of gene expression prokaryotic operons eukaryotic chromatin transcription factors post-transcriptional control  Online resources Other animations and video tutorials DNA learning centre – Cold Spring Harbours online DNA learning resource - https://dnalc.cshl.edu/websites/ DNA from the beginning – An animated primer of 75 experiments that made modern genetics http://www.dnaftb.org/ Bozeman Science basic video tutorial, Transcription and translation http://youtu.be/h3b9ArupXZg