Transcript Slide 1
1 What are needed for translation Permission Template (mRNA) Building blocks (20 types of aa) Ribosome tRNA Enzymes Energy (ATP & GTP) Protein factors 2 Different stages of Gene expression 3 Translation: The required materials 4 Translation: The Permission 5’ UTR 3’ UTR 5 The template: mRNA codons 1- mRNA always is read in 5’ to 3’ direction 2- The information for the translation are in the form of codon 3- Since there are 4 letters- A, C, U, G- the total number of codons is 64 4- 61 codon specify an aa the remaining UAA Ochre (UAA, UGA, UGA) are stop codons UAG Amber 5- The initiation codon is AUG which codes for Met UGA opal Codons: 1- are triplet 2- are almost Universal UUA 3- are unambiguous UUG 4-have no punctuation CUU The degeneracy CUC 5- are non-Overlapping CUA 6-are degenerate ( synonym) CUG 6 Open reading frame of mRNA One mRNA can be read in 3 different ways( reading frames) mRNA 5’--------AUGCAUGCAUGCGGU----------3’ 5’--------AUA GAU CCA UGA GGU----------3’ Blocked 5’-------A UAG AUC CAU GAG GU----------3’ Blocked 5’--------AU AGA UCC AUG AGG U----------3’ ORF( Open reading frame) * Only one is usually correct- gives the correct protein * Translation of the correct reading frame requires selection 7 8 Ribosome function subunits translation L composition sites S A Proteins types function str Pro 50 S& L Eu 80 S& L cat P E rRNA types genes repeated Pro 5 16 23 function Eu str 5.8 5 18 28 genes repeated cat ribozyme) 9 Ribosome: structure, subunits, sites, types •Is a ribonucleoprotein structure •Has two subunits •Has three sites (A, P & E) •May be 70 or 80 S 10 Ribosomal RNA 1- 60% ribosome is rRNA 2- rRNA has a complex secondary structure 3- various ribosomal proteins are associated with discrete areas of the rRNA 11 tRNA function aa carrier structure primary secondary tertiary trifolium arms aa TΨC Anti codon genes types repeated about 30 DHU L-shape is charged by aa- tRNA synthetases 12 Formation of aminoacyl tRNA (“charged tRNA”) 13 Enzymes 1- Amino acyl- tRNA Synthetases Linkage of aa the tRNAs 2- Peptidyle Transferase Peptidyle bond formation 3- Peptidyle Translocase Peptidyle Translocation from A-site to the P-site 14 Translation Factors of prokaryotes 15 16 Different Stages in Protein Synthesis Recognition & Initiation Elongation Termination ( Release) 17 How does the ribosome know where to begin protein synthesis ?– the recognition of ORF In Prokaryotes: ‘Shine-delgarno ’ is a 5-10 nt. sequence at 3’ 16S rRNA that is complementary to 5’ mRNA sequence just upstream of strat codon There is one SD for each cistron SD anchors ribosome to the start site Small changes in this sequence reduce the efficacy of the translation 18 How does the ribosome know where to begin protein synthesis?- initiation aa Most proteins begin on AUG (GUG & UUG less frequently) The first amino acid incorporated (in prokaryotes) is Formyl Methionine bound to an initiator tRNAfmet O R (amino acid side chain) HC-NH-CH-COOH - Note: f-Met looks like a peptide! - normal tRNAmet only recognizes AUG while tRNAf-Met will recognize AUG, GUG & UUG codons - f-Met only used at initiation 19 Recognition and Initiation in prokaryotes 20 Elongation in prokaryotes The new tRNA with next aa comes into the A site The formation of the peptide bond is catalyzed by the rRNA in the 50S large subunit ( ribozyme) 1- After formation of the peptide bond the ribosome is ‘translocated’ or moved down the RNA 2- The tRNA which now has the nascent peptide is now moved to the P site The new tRNA with next aa comes into the A site The tRNA that was previously attached to the peptide is then moved to the 21 E site, then released Summary of different stages of Translation in Prokaryotes Initiation Elongation Termination 22 23 Recognition and Initiation in Eukaryotes Most steps are the same, However the initiation is more complex More initiation factors( at least 11) are required No shine-Dalgarno in mRNA CAP binding factor recognize the 5’ end and by scanning the first AUG is find The initiator tRNA (tRNAi Met) is special but not formylated There are UTRs at the both ends of mRNA that regulate the translation rate IRES ( internal ribosomal entry sites) 24 Recognition and initiation in eukaryotes Activation of IFs by phosphorylation 25 Elongation & Termination in Eukaryotes Elongation is similar to Prokaryotes EF1B homolog to EF-Ts EF2 homolog to EF-G EF1A homolog to EF-Tu Termination even is simpler: Only one RF binds with GTP at the termination codon 26 Cell compartments and Protein sorting 27 Protein sorting cytosol nuclear envelope smooth ER lysosomes nucleus rough ER peroxisomes mitochondria Golgi plasma membrane secreted 28 Protein Sorting Requirements Compartments Stages Importance • Translation ER PM Gol …. • Recognition • Modification •Translocation mRNA- Signals SRP SRP Docking Leader receptor protein sequence cleaved Signal Energy Peptidase not cleaved 29 Protein Sorting machinery 30 Internal signal peptides of membrane proteins 31 Summary of Prokaryotic vs. Eukaryotic Translation 1- general processes are very similar 2- eukaryotic ribosomes are larger. 4.2 MDa vs 2.7 MDa 3- in Eukaryotes, special Meti tRNA is NOT formylated 4- No Shine-Delgarno sequence. Initiation occurs at the first start codon after 5’-cap 5 - Additional initiation factors required in eukaryotes 32