Translation Dr. Rajveer - PowerPoint Presentation

1. TranslationDr. Rajveer Singh ChauhanAssistant Professor,Department of Botany,DDU Gorakhpur University, Gorakhpur, Uttar Pradesh

2. Messenger RNA functions as the template for protein synthesis. It carries genetic information from DNA to ribosome and helps to assemble amino acid in their correct order.Both prokaryotes and eukaryotes contain three primary regions in the mRNA-5' UTRCoding region3' UTR5' UTR of prokaryotes consist of a consensus sequence called Shine- Dalgarno Sequence, serves as ribosome binding site during translation.Translation involves a change in language from nucleotide sequence of mRNA to amino acid sequence of polypeptide chain.Introduction

3. The cellular machinery for translated mRNA into peptide involves 5 major components-RibosomeTransfer RNA moleculesAmino acyl tRNA synthetasesMessenger RNA moleculesTranslation factorsTranscription and translation are coupled in prokaryotes while these events are separate in both time and space in eukaryotic cells.Each tRNA binds to one specific amino acid and each recognizes one or more mRNA codons that specify that particular amino acid as indicated by genetic code.The key of specificity between amino acid and its tRNA is a set of enzyme called amino acyl tRNA synthetases.

4. The recognition of tRNA by a synthetase depends on the differing nucleotide sequences of tRNA.The overall fidelity of translation depends on the accuracy of two processes: Codon -anticodon recognition Amino acylated tRNA synthesisAmino acyl tRNA are synthesized by 3' esterification of tRNA with the appropriate amino acid and uncharged tRNA is amino acylated to generate a charged tRNA which then interacts with a translation elongation factor.Amino acyl tRNA synthetase posses a variety of proofreading activities that serve to hydrolyse the mis-matched amino acid either before or after transfer to tRNA.

5. The proof-reading mechanism involves water mediated hydrolysis of mis-charged tRNA. The amino acyl tRNA synthetase displays an overall error rate about 1 in 10000.Translation can be divided into 3 main stages-InitiationElongationTerminationEach stage of protein synthesis involves multiple accessory factors and energy from ATP and GTP.

6. In this step all the components necessary for protein synthesis assemble:mRNASmall and large subunits of ribosomeInitiation factorsInitiator tRNAGTP.Initiation is the most complex and most tightly controlled of the steps in protein synthesis. A functional ribosome exist as 2 subunits- large and small. mRNA can bind to the small ribosomal subunit when subunits are separated.In prokaryotes, 3 initiation factors are involved – IF1, IF2, IF3. IF3 binds to small subunit of ribosome and prevents the large subunit from binding during initiation.Translation Initiation in Prokaryotes

7. Translation always start at a specific initiation codon . In bacteria , AUG is the initiation codon about 90% of time, GUG about 10%, UUG about 1%.In very rare cases other codons such as AUU may functions as the initiation codon.IF1, IF2-GTP and IF3 are needed for polypeptide chain initiation at physiological Mg++ concentration.High Mg++ stabilizes the 70S complex and permits mRNA and amino acyl tRNA molecule to bind directly to 70S ribosome within the ribosome binding site (Shine - Dalgarno Sequence = SDS) which is complementary to the 16S rRNA.

8. During the initiation the nucleotide in the SDS pair with anti-SDS allowing the small subunit of the ribosome to attach the mRNA and position mRNA directly over the initiation codon.After this the initiator tRNA attaches to the initiation codon. This step requires IF2 which forms the complex with GTP. The resulting components are known as 30S initiation complex.In the final step of initiation IF3 dissociate from the small subunit allowing the large subunit of ribosome to join the 30S initiation complex.The molecule of GTP is hydrolysed to GDP and IF1 and IF2 depart when the large sub-unit joins the 30S initiation complex. It is called 70S initiation complex.

9. Initiation complex Formation

10. Binding of Aminoacyl tRNARobinson, A. and Antoine Oijen, M. V. Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies . Nat. Rev. Microbiology 2013 (3); 1-13

11. TranslocationPost Translocation Pathway (2-3-2)

12. Peptide bond formation starts with initiator tRNA at the ribosomal P site and an empty A site.The elongation factor EF-TU forms a ternary complex with tRNA and GTP and carries the amino acylated tRNA to the vacant A site of the ribosome.The codon anticodon of mRNA and tRNA induces conformational changes in the small ribosomal subunit and 16S rRNA of small subunit (18S rRNA in eukayotes) interacts with the mRNA – tRNA duplex. This interaction activates the hydrolysis of GTP to GDP by EF-TU in bacteria. Elongation

13. The N-formylmethionine is liberated from the initiator tRNA and forms a peptide bond with its carboxyl group and the free amino group of the amino acid in the A site resulting in a tRNA with 2 attached amino acids. This peptidyl transferase reaction is carried out in the peptidyl transferase centre of the large ribosomal subunit and requires the co-ordinated action of various ribosomal proteins as well as ribosomal RNA (28S rRNA in eukaryotes, 23S rRNA in prokaryotes).

14. In the next step GTP bound EF-G in bacteria releases the deacylated tRNA from the E site and facilitates the transfer of the peptidyl tRNA from A to the site P site leaving a free A site for the next round of peptidyl transfer.This step is called translocation. Because the peptidyl tRNA translocated from A to P site and ribosome moves a codon forward on mRNA.This cycle is repeated until a stop codon encountered and termination process is initiated.

15. Translation is terminated as a consequence of a presence of a stop codon in A site.There are no tRNA with anticodons complementary to the termination codon.No tRNA enters the A site of ribosome when a termination codon is encountered, instead release factor bind to ribosome.Release factors that are present on ribosome recognize stop codon and facilitate the release of polypeptide chain. (class 1 release factor)Class 2 release factors are GTPases and stimulate the activity of class 1 release factor.Termination

16. In bacteria class 1 RFs are RF1 and RF2 decode the stop codon UAA whereas UAG is decoded by RF1 and UGA by RF2 only.RF3 is the class 2 release factor in bacteria that activates class1 release factors.Eukaryotes posses a single release factor eRF1 that decodes all of the three stop codons.The class 2 RF eRF3 is required for eRF1 function.Either RF1 or RF2 binds at a termination codon, it induces peptidyl transferases to transfer the growing polypeptide chain to a water molecule rather than to another amino acid.

17. A overview of Translation TerminationSteitz, T. A. A structural understanding of the dynamic ribosome machine. Nat. Rev. Mol. Cell Bio. 2008 (9); 242-253

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