Part 2 Module 4 Translation and regulation of gene expression PDF

Module-4 Part 2 Translation and regulation of gene expression Course taken by Dr. Neha deora Assistant Professor Dayanand...

Module-4 Part 2 Translation and regulation of gene expression Course taken by Dr. Neha deora Assistant Professor Dayananda Sagar University  Topics covered in Module 4 1. Genetic code, wobble hypothesis, Mechanism of translation in prokaryotes and eukaryotes 2. Post translational modification of Proteins. Regulation of Gene expression in Prokaryotes –Operon concepts, induction, repression, attenuation, examples of Lac and Trp operons 2. Regulation of Gene expression in Eukaryotes –galactose metabolism in yeast.  Wobble hypothesis Francis and Nick explained the Wobble hypothesis in 1966 to explain the flexibility in the base pairing between the third nucleotide of a codon in (mRNA) and the corresponding anticodon in tRNA. According to the Wobble Hypothesis, only the first two bases of the codon have a precise pairing with the bases of the anticodon of tRNA (the first (5′) base of the codon pairs with the third (3′) base of the anticodon), while the third bases can tolerate some flexibility in pairing or may Wobble. The flexibility at the third position means that a single tRNA can recognize multiple codons that code for the same amino acid. This reduces the number of tRNAs needed to translate the genetic code. For example, alanine is encoded by the codons GCU, GCC, GCA, and GCG, all of which begin with GC and these codons differ only at 3rd position. tRNA with anticodon CG could pair with codons at first and second position, while allowing some flexibility at the third position.  They proposed that there could be some nonstandard pairings of bases at the third position of a codon. For instance, Inosine (which is found in the anticodon of some tRNAs) can pair with A, U, or C, allowing one tRNA to recognize several codons. The anticodon 5'-AIA-3' (where I represent inosine) can pair with the codons 5'-UAU-3', 5'-UAC-3', or 5'-UAA-3’. The wobble hypothesis explains how the genetic code can be efficiently read with a relatively small number of tRNAs. Instead of needing 61 different tRNAs for the 61 codons (since there are 64 possible codons), wobble pairing allows for fewer tRNAs to cover all codons, since some tRNAs can recognize multiple codons that encode the same amino acid. This allows for multiple codons to be recognized by a single tRNA, and thereby reducing the number of tRNAs required to read the genetic code. This contributes to the efficiency of protein synthesis.  Translation in prokaryotes Protein synthesis is divided into four stages: (1) tRNA charging, which entails the binding of amino acids to the tRNAs (2) Initiation, in which the components necessary for translation are assembled at the ribosome (3) Elongation, in which amino acids are joined, one at a time, to the growing polypeptide chain (4) Termination, in which protein synthesis halts at the termination codon and the translation components are released from the ribosome.  tRNA charging Binding of amino acid to tRNA molecule is called tRNA charging. Each tRNA is specific for only one amino acid. The key to specificity between an amino acid and its tRNA is a set of enzymes called aminoacyl-tRNA synthetases. A cell has 20 different aminoacyl-tRNA synthetases, one for each of the 20 amino acids. Each synthetase recognizes a particular amino acid, as well as all the tRNAs that accept that amino acid. tRNA charging requires energy, which is supplied by adenosine triphosphate (ATP) The carboxyl group (COO−) of the amino acid is attached to the adenine nucleotide at the 3′ end of the tRNA.  Overall reaction amino acid + tRNA + ATP → aminoacyl-tRNA + AMP + PPi Structure of tRNA Initiation of translation At this stage, all the components necessary for protein synthesis assemble : mRNA, the small and large subunits of the ribosome, a set of proteins called initiation factors, initiator tRNA with N-formyl methionine attached, and guanosine triphosphate (GTP). Initiation comprises three major steps. Binding of to the small subunit of the ribosome. Binding of initiator tRNA to the mRNA through base pairing between the codon and the anticodon. Formation of initiation complex through joining of the large ribosome. The functional ribosome of prokaryotes exists as two subunits, the small 30S subunit and the large 50S subunit. When not actively translating, the two subunits are joined An mRNA molecule can bind to the small ribosome subunit only when the subunits are separate.  Initiation factor 3 (IF-3) binds to the small subunit of the ribosome and prevents the large subunit from binding during initiation. Another factor, initiation factor 1 (IF-1), enhances the disassociation of the large and small ribosomal subunits. Next, the initiator tRNA, fMet-tRNA, attaches to the initiation codon. This step requires initiation factor 2 (IF-2), which forms a complex with GTP. At this point, the initiation complex consists of: a small subunit of the ribosome, the mRNA, the initiator tRNA with its amino acid one molecule of GTP, and several initiation factors. These components are collectively known as the 30S initiation complex. In the final step of initiation, initiation factors disassociate from the small subunit, allowing the large subunit of the ribosome to join the initiation complex. When the large subunit has joined the initiation complex, the complex is called the 70S initiation complex.  mRNA for translation The 5′ untranslated region (5′ UTR; sometimes called the leader), a sequence of nucleotides at the 5′ end of the mRNA, does not encode any of the amino acids of a protein. In bacterial mRNA, this region contains a consensus sequence called the Shine–Dalgarno sequence, which serves as the ribosome- binding site during translation; it is found approximately seven nucleotides upstream of the called the start codon. The next section of mRNA is the protein-coding region, which comprises the codons that specify the amino acid sequence of the protein. The protein-coding region begins with a start codon and ends with a stop codon. The last region of mRNA is the 3′ untranslated region (3′ UTR; sometimes called a trailer), a sequence of nucleotides that is at the 3′ end of the mRNA and not translated into protein. The 3′ untranslated region is required for the stability of mRNA and the translation of the mRNA protein-coding sequence.