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Translation.Pdf TRANSLATION Course Content - i. Mechanism of Protein Synthesis ii. Protein Sorting iii. Inhibitors of Protein Synthesis iv. Post -translational Modifications v. HSPs Gurumayum Suraj Sharma THREE BASIC STAGES OF PROTEIN SYNTHESIS I. Initiation II. Elongation III. Termination Similar in bacteria & eukaryotes. Various Proteins & Factors Involved in Translation Gurumayum Suraj Sharma INITIATION OF TRANSLATION INITIATION Encompasses all steps preceding formation of the peptide bond between the first two amino acids in the polypeptide chain. Initiation involves an mRNA MOLECULE , a RIBOSOME , a SPECIFIC INITIATOR tRNA , PROTEIN INITIATION FACTORS (IF) & GTP (GUANOSINE TRIPHOSPHATE). Gurumayum Suraj Sharma INITIATION - BACTERIA First step in initiation - Interaction of 30S [small] ribosomal subunit to which IF -1 & IF -3 are bound with region of mRNA containing AUG initiation codon . IF -3 aids in binding of subunit to mRNA & prevents binding of 50S ribosomal subunit to 30S subunit. AUG initiation codon alone not sufficient to indicate where 30S subunit should bind to mRNA A sequence upstream [to 5’ side in leader of mRNA] of AUG called Ribosome-binding site [RBS] also needed. Gurumayum Suraj Sharma John Shine & Lynn Dalgarno [1970s] hypothesized that purine -rich RBS sequence (5’-AGGAG -3’ or some similar sequence) & sometimes other nucleotides in this region could pair with a complementary pyrimidine- rich region [5’-UCCUCC -3’] at 3’ end of 16S rRNA . Shine-Dalgarno Sequence Gurumayum Suraj Sharma Joan Steitz First to demonstrate pairing experimentally. The mRNA RBS region is now commonly known as the Shine - Dalgarno sequence. Sequences Involved in Binding of Ribosomes to mRNA in Initiation of Protein Synthesis in Prokaryotes. Gurumayum Suraj Sharma Most RBSs are 8-12 nucleotides upstream from initiation codon. MODEL - Formation of complementary bp between mRNA & 16S rRNA allows small ribosomal subunit to locate true sequence in mRNA for initiation. GENETIC EVIDENCE If Shine-Dalgarno sequence mutated - Its possible pairing with 16S rRNA sequence significantly diminished or prevented, mutated mRNA cannot be translated. Likewise, if rRNA sequence complementary to Shine–Dalgarno sequence is mutated , mRNA translation cannot occur. Loss of translatability - A result of mutations in one or other RNA partner could be caused by effects unrelated to the loss of pairing of the two RNA segments, a more elegant experiment was done. Mutations were made in the Shine-Dalgarno sequence to abolish pairing with wild-type rRNA sequence, and compensating mutations were made in the rRNA sequence so that the two mutated sequences could pair. In this case, mRNA translation occurred normally, indicating the importance of the pairing of the two RNA segments. Gurumayum Suraj Sharma Next step in initiation Binding of a special initiator tRNA to AUG start codon to which 30S subunit is bound. In both prokaryotes and eukaryotes AUG initiator codon specifies methionine. Thus, newly made proteins in both types of organisms begin with methionine. Methionine may be removed later in many cases. Gurumayum Suraj Sharma BACTERIA - Initiator tRNA is tRNA.fMet [with anticodon 5’-CAU -3’] bind to AUG start codon. This tRNA carries a modified form of methionine, formylmethionine (fMet) Formyl group added to amino group of methionine. First, methionyl -tRNA synthetase catalyzes addition of methionine to tRNA. Then enzyme TRANSFORMYLASE adds formyl group to methionine. The resulting molecule is designated fMet -tRNA When AUG codon in mRNA molecule is encountered at a position other than start of amino acid-coding sequence Different tRNA [called tRNA.Met], is used to insert methionine at that point in polypeptide chain. Gurumayum Suraj Sharma Initiator tRNA , fMet - tRNA.fMet , is brought to 30S subunit-mRNA complex by IF - 2, which also carries a molecule of GTP. Initiator tRNA binds to subunit in P site. All aminoacyl-tRNAs that come to ribosome bind to A site . However, IF-1 bound to 30S subunit is blocking A site so that only P site is available for initiator tRNA to bind to. Thus, 30S initiation complex formed Consists of mRNA, 30S subunit, initiator tRNA, and the initiation factors. Next, 50S ribosomal subunit binds, leading to GTP hydrolysis and release of three initiation factors. The final complex is called the 70S initiation complex. Gurumayum Suraj Sharma INITIATION IN EUKARYOTES Similar in eukaryotes, however, more complex & involves many more initiation factors, called eukaryotic initiation factors [eIF] MAIN DIFFERENCES Initiator methionine is unmodified , although a special initiator tRNA still brings it to the ribosome Shine-Dalgarno sequences are not found in eukaryotic mRNAs. Instead, eukaryotic ribosome uses another way to find AUG initiation codon. First, a eukaryotic initiator factor eIF-4F- A multimer of several proteins, including eIF-4E, the capbinding protein [CBP] - Binds to cap at 5’ end of mRNA. Then, a complex of 40S ribosomal subunit with initiator Met- tRNA, several eIF proteins, & GTP binds, together with other eIFs, & moves along mRNA, scanning for initiator AUG codon. AUG codon is embedded in a short sequence- called Kozak sequence [after Marilyn Kozak ], indicates that it is initiator codon. - Process is called scanning model for initiation. Gurumayum Suraj Sharma AUG codon is almost always the first AUG codon from 5’ end of mRNA But, to be an initiator codon, it must be in an appropriate sequence context. Once 40S subunit finds AUG, it binds to it, and then 60S ribosomal subunit binds, displacing eIFs (except for eIF-4F, which is needed for the subsequent initiation of translation ), producing 80S initiation complex with initiator Met-tRNA bound to mRNA in P site of ribosome. Poly(A) tail of eukaryotic mRNA also plays a role in translation . Poly(A) Binding Protein II [PABPII] bound to poly(A) tail also binds eIF-4G [one of the proteins of eIF-4F at cap] , thereby looping the 3’ end of mRNA close to 5’ end. Thus, poly(A) tail stimulates initiation of translation . Gurumayum Suraj Sharma Initiation of protein synthesis: A 30S ribosomal subunit, mRNA, initiator fMet-tRNA, and initiation factors form a 30S initiation complex. Next, the 50S ribosomal subunit binds, forming a 70S initiation complex. During this event, the initiation factors are released and GTP is hydrolyzed. Gurumayum Suraj Sharma ELONGATION OF POLYPEPTIDE CHAIN After initiation is complete, the next stage is elongation. The addition of amino acids to the growing polypeptide chain one by one- as they take place in bacteria. THREE STEPS : 1. Aminoacyl -tRNA (charged tRNA) binds to ribosome in A site. 2. A peptide bond forms. 3. Ribosome moves (Translocates) along mRNA one codon. As with initiation, elongation requires accessory protein factors, called elongation factors [EF] , and GTP. Elongation is similar in eukaryotes Gurumayum Suraj Sharma Binding of Aminoacyl -tRNA At start of elongation, anticodon of fMet -tRNA is H-bonded to AUG initiation codon in P site of the ribosome. Next codon in mRNA is in A site Next, the appropriate aminoacyl-tRNA binds to codon in A site. This aminoacyl-tRNA is brought to ribosome bound to EF -Tu -GTP [a complex of the protein elongation elongation factor, EF- G]. Gurumayum Suraj Sharma An EF-G-GTP complex binds to ribosome, GTP is hydrolyzed, and translocation of ribosome occurs along with displacement of uncharged tRNA away from P site. It is possible that GTP hydrolysis changes the structure of EF-G, which facilitates translocation event. Gurumayum Suraj Sharma Translocation similar in eukaryotes - Elongation factor in this case is eEF-2, which functions like bacterial EF-G. Uncharged tRNA moves from P site and then binds transiently to E site in 50S ribosomal subunit, blocking next aminoacyl-tRNA from binding to A site until translocation is complete & peptidyl-tRNA is bound correctly in P site. Once that has occurred, uncharged tRNA is then released from ribosome. After translocation, EF-G is released and then reused. Gurumayum Suraj Sharma During translocation , peptidyl-tRNA remains attached to its codon on mRNA Since ribosome has moved, peptidyl-tRNA is now located in the P site (hence name peptidyl site ). After completion of translocation, A site is vacant. An aminoacyl–tRNA with correct anticodon binds to newly exposed codon in A site, reiterating the process already described. The whole process is repeated until translation terminates at a stop codon. Gurumayum Suraj Sharma Once ribosome moves away from initiation site on mRNA, another initiation event occurs [occurs both in Prokaryotes & Eukaryotes]. The process is repeated until, typically, several ribosomes are translating each mRNA simultaneously. The complex between an mRNA molecule and all the ribosomes that are translating it simultaneously is called a polyribosome, or polysome. Each ribosome in a polysome translates the entire mRNA and produces a single, complete polypeptide. Polyribosomes enable a large number of polypeptides to be produced quickly and efficiently from a single mRNA. Polysome- A number of ribosomes, each translating same mRNA sequentially TERMINATION OF TRANSLATION Termination - Signalled by one of three stop codons (UAG, UAA, and UGA), which are the same in prokaryotes and eukaryotes. Stop codons do not code for any amino acid, so no tRNAs in cell have anticodons for them. Ribosome recognizes a stop codon with the help of proteins called RELEASE FACTORS [RF] , which
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