1

Protein Synthesis Dr. Ishtiaq Ahmad Khan

Information transmission Information stored in DNA transferred to RNA and then expressed in the structure of proteins – Two steps in process: Transcription - information transcribed from DNA into mRNA Translation - information in mRNA translated into primary sequence of a protein

tRNA Carries amino acid at 3΄-end Contains anti-codon to recognize mRNA codon Physical link between the nucleotide sequence of nucleic acids and the amino acid sequence of proteins 4

5

Structural Features Length nucleotides the hydrogen-bonding pattern of all tRNAs forms a cloverleaf structure with four arms; the longer tRNAs have a short fifth arm, or extra arm. The amino acid arm can carry a specific amino acid esterified by its carboxyl group to the 2- or 3- hydroxyl group of the A residue at the 3 end of the tRNA. The anticodon arm contains the anticodon The other major arms are the D arm, which contains the unusual nucleotide dihydrouridine (D) TψC arm, which contains ribothymidine (T), not usually present in RNAs, and pseudouridine (ψ), which has an unusual carbon–carbon bond between the base and ribose 6

The D and TψC arms contribute important interactions for the overall folding of tRNA molecules, and the Tψ C arm interacts with the large-subunit rRNA. 7

Modified Bases 8 IInocine mImethylinosine mGmethylguanosine m2Gm2Gdimethylguanosine ψPseudouridine DDihydrouridine

9 Normal Bases

10 Modified Bases in tRNA

11

12

13 The N-7, O 6, and N 6 of purines, the atoms that participate in the hydrogen bonding of triplex DNA, are often referred to as Hoogsteen positions, and the non-Watson- Crick pairing is called Hoogsteen pairing,

14

Suppressor tRNA When a mutation introduces a termination codon in the interior of a gene, translation is prematurely halted and the incomplete polypeptide is usually inactive. These are called nonsense mutations. The gene can be restored to normal function if a second mutation either (1)converts the misplaced termination codon to a codon specifying an amino acid (2)Suppresses the effects of the termination codon. Such restorative mutations are called nonsense suppressors; they generally involve mutations in tRNA genes to produce altered (suppressor) tRNAs that can recognize the termination codon and insert an amino acid at that position. Most known suppressor tRNAs have single base substitutions in their anticodons. 15

The genetic code is nearly universal Genetic Code

17 Amber Suppressor

18 Ocher Suppressor

Translation - animation

Translation (protein synthesis) Information in mRNA translated into protein in 5 steps: – ACTIVATION – INITIATION – ELONGATION – TERMINATION & – Folding and Post translational modifications

Translation (protein synthesis) ACTIVATION – Each amino acid activated by reacting with ATP – tRNA synthetase enzyme attaches activated amino acid to own particular tRNA Adapted from: Bettelheim FA and March J (1990) Introduction to Organic and Biochemistry (International Edition). Philadelphia: Saunders College Publishing p398

Initiation complex in eukaryotes

Termination UAA UAG UGA Termination or release factors

Termination cont….. In bacteria, once a termination codon occupies the ribosomal A site, three termination factors, or release factors—the proteins RF-1, RF-2, and RF-3— contribute to ( 1) hydrolysis of the terminal peptidyltRNA bond; (2) release of the free polypeptide and the last tRNA, now uncharged, from the P site (3) dissociation of the 70S ribosome into its 30S and 50S subunits, ready to start a new cycle of polypeptide synthesis

RF-1 recognizes the termination codons UAG and UAA, and RF-2 recognizes UGA and UAA. Either RF-1 or RF-2 (depending on which codon is present) binds at a termination codon and induces peptidyl transferase to transfer the growing polypeptide to a water molecule rather than to another amino acid.

Thanks