• Protein synthesis “Translation”

1. • Protein synthesis “Translation” • The letters of the nucleic acid is translated into amino acids. “from nucleotide language to amino acid language” • • Genes specify the amino acids sequence in proteins. • Genetic code: the relation between the sequence of bases in DNA (or it’s transcript RNA) and the sequences of amino acid in protein. • • Features of Genetic Code • - Coding ratio (3 base-code) • - We have 4 bases and 20 amino acid: • Single-base code = 4 • Two-base code = 4 * 4 = 16 • Three-base code = 4 * 4 * 4 = 64 • Three-base code • More than code can specify one amino acid

2. An amino acid is coded by three bases called “codon” and these condones: - Non-overlapping - The sequence of bases is read sequentially from a fixed starting point. - There are no commas between these triplets.

3. • The genetic code is specific • Specific codon always codes for the same amino acid • • Redundant • - For a given amino acid may have more than one codon for it. • - Codons that specify the amino acid are called “synonyms” most of them differ only in the last base of the triplet • UUU • UUC • • Universal • The genetic code almost universal in the whole of prokaryotic, plant, animal kingdoms, the same codon used for the same amino acid. • With few exceptions: like in the mitochondria • Codon Common code Mitochondrial code • AUC Ile Met • AGAArg STOP • AGGArg STOP • UGASTOP Trp phe

4. START AUG STOP UAA , UAG UGA UC AG UC AG UCAGUC AG Third letter of codon

5. • Consequences of altering the nucleotide sequence “mutation” • Base substitution “Point mutation” • - Changing a single nucleotide base on the m-RNA chain, and this can lead to: • Silent mutation • The codon containing the changed base codes for the same amino acid • UCA silent UCU • Serine Serine • 2. Missense • The change results in a new different amino acid • UCA missense CCA • Serine Proline • Non-sense mutation • The change leads to premature termination if the codon containing the changed base become a termination codon. • UCA non-sense UAA • Serine STOP codon

6. Base Deletion or Insertion • Frame shift mutation • Insertion or deletion of one or two bases will alter the reading frame and this cause extensive change in the translated protein absolutely different protein • Insertion or deletion of one codon “3 nucleotides” • This lead to addition of new amino acids (if three bases were inserted), or to deletion of one amino acid (if three bases were deleted). • The reading frame in this case is not changed and the produced protein is not extensively changed.

7. Missense Mutation

8. Non-Sense Mutation

9. • The Major Participants in Translation • A large number of components are required for the synthesis of polypeptides • Amino acids: absence of 1 amino acid  termination of the polypeptide at that amino acid • m-RNA:act as template for protein synthesis. • t-RNA:adaptors • Functional Ribosomes:protein synthesis machine. • Energy sources • Translation factors • Enzymes • - The translation takes place in the cytosol • • t-RNA • - At least one specific t-RNA is required for each amino acid. In human there are 50 types of tRNA and in prokaryotes there are 30 – 40 tRNA • - 20 amino acid  more than tRNA type for a given amino acid • - tRNA has uncommon and modified bases (Inosine, Pseudouracil, … ) • - All tRNA types have a common structure

10. • tRNA structure • - Two functional parts • Acceptor stem (amino acid attachment site) • 3’-terminus of tRNA has always the sequence 5’ … CCA-OH 3’ • Anti codon • Three base nucleotide sequence. That recognize a specific codon on the mRNA and they are complementary and anti parallel, the codon specifies the amino acid that will be inserted into the growing polypeptide.

11. tRNA Structure

12. • Codon Recognition by tRNA • - Recognition of a codon in the mRNA is accomplished by anti codon sequence of the tRNA • - Some tRNA can recognize more than codon • - Anti codon + codon binding follows the complementary and anti parallel binding • • Wobble hypothesis • - The base at the 5’- end of anti codon is not spatially defined and this allows non-traditional base pairing with the 3’- base of the codon. • - The result of wobbling is that there need not be 61 tRNA types to read the 61 codons that code for the amino acids Wobble position Anti codon3’…UAC…5’ Codon 5’ …AUG…3’ Anti codon5’ …CAU…3’

13. •Coupling of tRNA to amino acids - Amino acids are covalently attached to OH group of the ribose sugar of the adenosine residue at the 3’- end of tRNA. - Each aminoacyl tRNA synthestase recognizes a specific amino acid and the tRNAs that correspond to that amino acid. - These enzymes are highly specific tRNA – amino acid = activated amino acid or charged tRNA.

14. • Ribosomes • Machines for protein synthesis. • - rRNA – protein complex • - Major cell constituents, an E. coli contains 15000 ribosomes forming 25% of the dried cell • - In eukaryotic cell the ribosomes either free in the cytosol or in close association with endoplasmic reticulum (ER) • - Mitochondria contains their own set of ribosomes.

15. • Ribosomal proteins • - These proteins play important roles in the structure and function of the ribosome.

16. • The Mechanism of Translation • The pathway of protein synthesis is called translation. • Because the language of nucleotides of the mRNA is transcripted into amino acid language. • - The mRNA is translated in 5’  3’ direction producing polypeptide from it’s amino terminal end to its carboxylic terminus. • - One prokaryotic mRNA can code for different polypeptide types (poly cistronic). Because m-RNA contains different coding regions with different initiators. 5’ 3’ AUG UAA AUG UAG Code for protein A Code for protein B Each eukaryotic mRNA code only for one polypeptide (mono cistronic)

17. GTP • •Steps in protein synthesis • Initiation The small ribosomal subunit (Shine-Dalgarno sequence) Formyl group is added to the charged tRNA met by the enzyme transformylase (formyl THF is the source) Will be Met in eukaryotes The release of IF3 increase the affinity to the large ribosomal subunit The formyl group will be removed during the elongation The Met amino acid will be cleaved from the polypeptide. Specifies the next a.a

18. • The binding of mRNA to 30 S ribosomal subunit The 16S rRNA has a nucleotide sequence near it’s 3’ – end that complementary to Shine-Dalgarno sequence(nucleotide bases 5’ – UAAGGAGG – 3’ located 6 – 10 bases up stream to the AUG codon on the mRNA) - The mRNA 5’- end and 3’- end of rRNA (in the 30S ribosomal subunit) can form complementary base pair and this can facilitate the binding of the mRNA to 30S ribosomal unit.

19. Elongation The addition of a.a to the carboxyl end of the growing polypeptide chain. The delivery of a.a - tRNA to A site Peptidyl transferase (integral part of 50S subunit) GTP

20. GTP Elongation This process will be repeated until a termination codon is reached. By each cycle the polypeptide has grown by one residue and consumed two GTP. Translocation: Moves by 3 nucleotides

21. GTP • Termination • RF1 recognizes UAA and UAG • RF2 recognizes UAA and UAG • RF3 is GTPase (stimulate the release process via GTP binding and hydrolysis) Termination codons UAA UAG UGA

24. •Polyribosomes (polysomes) Many ribosomes can simultaneously translate one mRNA.

25. • Energetic of translation - The energy cost for protein synthesis is high. - The total energy required for synthesizing a protein of N residues. 2N ATPs are required to charge tRNAs 1 GTP is needed for initiation. N –1 GTPs are needed to form N –1 peptide bonds N –1 GTPs are needed to form N –1 translocation steps 1 GTP is needed for termination So the total energy: 2N+1 + N-1 + N-1 + 1 = 4 N • Post translational modification. ”The final stage of protein synthesis” Folding and covalent modification. The produced protein may fold to form the 3° structure and may associate with other subunits. The covalent modification involve: Phosphorylation, Glycosylation, Hydroxylation Trimming

26. Protein synthesis “Translation”

27. The End GOOD LUCK

28. •Coupling of tRNA to amino acids - Amino acids are covalently attached to OH group of the ribose sugar of the adenosine residue at the 3’- end of tRNA. - Each aminoacyl tRNA synthestase recognizes a specific amino acid and the tRNAs that correspond to that amino acid. - These enzymes are highly specific tRNA – amino acid = activated amino acid or charged tRNA. Activation of the amino acid Aminoacyl-tRNA synthestase Adding the amino acid to the specific tRNA Formation of ester bond

29. The small ribosomal subunit • Steps in protein synthesis • Initiation (Shine-Dalgarno sequence) Formyl group is added to the charged tRNA met by the enzyme transformylase (formyl THF is the source) The release of IF3 increase the affinity to the large ribosomal subunit Will be Met in eukaryotes The formyl group will be removed during the elongation The Met amino acid will be cleaved from the polypeptide. Specifies the next a,a

30. The delivery of a.a - tRNA to A site Peptidyl transferase (integral part of 50S subunit) By each cycle the polypeptide has grown by one residue and consumed two GTP. This process will be repeated until a termination codon is reached. Moves with 3 nucleotides Translocation

31. Termination RF1 recognizes UAA and UAG RF2 recognizes UAA and UAG RF3 is GTPase (stimulate the release process via GTP binding and hydrolysis) UAA UAG UGA