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2 Permission Template (mRNA) Building blocks (20 types of aa) Ribosome tRNA Enzymes Energy (ATP & GTP) Protein factors What are needed for translation

3 Different stages of Gene expression

4 Translation: The required materials

5 Translation: The Permission 5 ’ UTR3 ’ UTR

6 The template: mRNA codons UUA UUG CUU CUC CUA CUG 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 codon specify an aa the remaining (UAA, UGA, UGA) are stop codons 5- The initiation codon is AUG which codes for Met Codons: 1- are triplet 2- are almost Universal 3- are unambiguous 4-have no punctuation 5- are non-Overlapping 6-are degenerate ( synonym) The degeneracy UAA Ochre UAG Amber UGA opal

7 One mRNA can be read in 3 different ways( reading frames ) * Only one is usually correct- gives the correct protein * Translation of the correct reading frame requires selection Open reading frame of mRNA mRNA 5’ AUGCAUGCAUGCGGU ’ 5’ AUA GAU CCA UGA GGU ’ Blocked 5’ A UAG AUC CAU GAG GU ’ Blocked 5’ AU AGA UCC AUG AGG U ’ ORF ( Open reading frame)

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9 Ribosome function subunits composition sites types function genes repeated Pro Eu str cat ribozyme) Pro Eu S& L 50 pro structural repeated 80 Eu catalytic str catrepeated translation L SA P E Proteins rRNA

10 Ribosome: structure, subunits, sites, types Is a ribonucleoprotein structure Has two subunits Has three sites (A, P & E) May be 70 or 80 S

% ribosome is rRNA 2- rRNA has a complex secondary structure 3- various ribosomal proteins are associated with discrete areas of the rRNA Ribosomal RNA

12 tRNA function structure arms genes types repeated about 30 aa carrier primary secondary tertiary aa TΨC Anti codon DHU trifolium L-shape is charged by aa- tRNA synthetases

13 Formation of aminoacyl tRNA (“charged tRNA”)

14 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

15 Translation Factors of prokaryotes

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17 Different Stages in Protein Synthesis R Recognition & Initiation E Elongation T Termination ( Release)

18 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 How does the ribosome know where to begin protein synthesis ?– the recognition of ORF

19 Most proteins begin on AUG (GUG & UUG less frequently) The first amino acid incorporated (in prokaryotes) is Formyl Methionine bound to an initiator tRNA fmet HC-NH-CH-COOH O R (amino acid side chain) How does the ribosome know where to begin protein synthesis?- initiation aa - Note: f-Met looks like a peptide! - normal tRNA met only recognizes AUG while tRNA f-Met will recognize AUG, GUG & UUG codons - f-Met only used at initiation

20 Recognition and Initiation in prokaryotes

21 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 E site, then released

22 Summary of different stages of Translation in Prokaryotes InitiationElongationTermination

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24 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 (tRNA i 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)

25 Recognition and initiation in eukaryotes Activation of IFs by phosphorylation

26 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

27 Cell compartments and Protein sorting

28 Protein sorting mitochondria rough ER peroxisomes cytosol nucleus plasma membrane Golgi lysosomes nuclear envelope smooth ER secreted

29 Protein Sorting RequirementsStages SignalsSRP SRP receptor mRNA- Leader sequence Docking protein Signal Peptidase Energy Translation Recognition Modification Translocation Compartments ER PM Gol …. cleaved not cleaved Importance

30 Protein Sorting machinery

31 Internal signal peptides of membrane proteins

32 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 Met i 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