Gene Expression: Protein Translation & Post-Translational Regulation - FHSC204

Chapter 5 Gene Expression: Protein Translation and post-translational regulation  Open reading frames (ORF)  Open reading frames are part of reading frames that do not contain stop codons  An open reading frame is the length of DNA or RNA through which the ribosome...

Chapter 5 Gene Expression: Protein Translation and post-translational regulation  Open reading frames (ORF)  Open reading frames are part of reading frames that do not contain stop codons  An open reading frame is the length of DNA or RNA through which the ribosome can move and add amino acids one after the other till it reaches a codon that does not code for any amino acid.  In simple words, an open reading frame is the region of the nucleotide sequence from the start codon to the stop codon  Necessary components for translation 1-messenger RNA  Encodes for specific protein sequence  Variable length ( depending on protein size)  Information is read in triplets (codons), 64 possible codons (4*4*4=64),61 are specific amino acids and 3 are terminator signals  Necessary components for translation 1-messenger RNA mRNA is complementary to DNA and read in triplets(codons)  Necessary components for translation 2-Transfer RNA(tRNA)  Bring one amino acid at a time to the growing polypeptide chain  Small molecule  Forms a cloverleaf structure  Anticodon: base pairs to mRNA codon during translation  Amino acid binding site: at 3’ end of the molecule  Necessary components for translation 2-Transfer RNA(tRNA) activation  The enzyme (aminoacyl-tRNA synthetase) first binds the amino acid to a molecule of ATP (to form an amino acid-AMP complex linked by a high- energy bond)  The amino acid is then transferred to the 3'-end of the appropriate tRNA, attaching to a terminal CCA sequence on the acceptor stem and releasing the AMP molecule  The tRNA molecule with an amino acid attached is thus said to be 'charged' and is now capable of participating in translation  The energy in the bond linking the tRNA molecule to the amino acid will be used in translation to form a peptide bond between adjacent amino acids  Necessary components for translation 3-Ribosomal RNA( rRNA)  Ribosome is the site of protein synthesis  Facilitates coupling of mRNA to tRNA  Huge molecule: small and large subunits should assemble for translation  Ribosome composition is 60% rRNA and 40% protein  Necessary components for translation 3-Ribosomal RNA( rRNA)  Steps of translation 1- Initiation 2- Elongation 3- Termination 1- Initiation Initiation of translation in prokaryotes The signal indicating where translation should begin on the mRNA is called ribosome binding site. Containing 2 elements: Shine-Dalgarno box The triplet AUG A special initiator tRNA, with a 5’-CAU-3’ anticodon, recognizes an AUG preceded by the Shine-Dalgarno box of a ribosome binding site. The initiator tRNA carries N-formylmethionine (fMet) Initiation of translation in Eukaryotes  The initiator tRNA–methionine complex (Met–tRNAi) is first loaded into the small ribosomal subunit along with additional proteins called eucaryotic initiation factors, or eIFs  Next, the small ribosomal subunit binds to the 5’ cap of mRNA molecule and its two bound initiation factors, eIF4E and eIF4G  The small ribosomal subunit then moves forward (5’ to 3’) along the mRNA, searching for the first AUG. Initiation of translation in Eukaryotes  In 90% of mRNAs, translation begins at the first AUG encountered by the small subunit. At this point, the initiation factors dissociate, allowing the large ribosomal subunit to assemble with the complex and complete the ribosome. The initiator tRNA is still bound to the P-site, leaving the A site vacant. Protein synthesis is therefore ready to begin 2- Elongation  Two elongation factors enter and leave the ribosome during each cycle, each hydrolyzing GTP to GDP and undergoing conformational changes in the process.  These factors are called EF-Tu and EF-G in bacteria, and EF1 and EF2 in eucaryotes.  EF-Tu simultaneously binds GTP and aminoacyl-tRNAs. In addition to helping move translation forward, EF-Tu (EF1 in eucaryotes) increases the accuracy of translation  EF-Tu checks whether the tRNA–amino acid match is correct  EF-Tu monitors the initial interaction between the anticodon of an incoming aminoacyl-tRNA and the codon of the mRNA in the A-site. 3-Termination  The end of the protein-coding message is signaled by the presence of one of three stop codons (UAA, UAG, or UGA).  Proteins known as release factors bind to any ribosome with a stop codon positioned in the A site, forcing the peptidyl transferase in the ribosome to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA.  This reaction frees the carboxyl end of the growing polypeptide chain from its attachment to a tRNA molecule, and since only this attachment normally holds the growing polypeptide to the ribosome, the completed protein chain is immediately released into the cytoplasm.  The ribosome then releases the mRNA and separates into the large and small subunits, which can assemble on this or another mRNA molecule to begin a new round of protein synthesis.  Monocistronic vs Polycistronic Polycistronic mRNA is a mRNA that encodes several proteins and is characteristic of many bacterial (prokaryotes) and chloroplast mRNAs Monocistronic mRNA is a mRNA that encodes only one protein and all eukaryotic mRNAs are monocistronic. Regulation of gene expression at translation level  Regulation of gene expression by eif2 Eukaryotic cells decrease their overall rate of protein synthesis in response to a variety of situations, including deprivation of growth factors or nutrients, infection by viruses, and sudden increases in temperature. Much of this decrease is caused by the phosphorylation of the translation initiation factor eIF2 by specific protein kinases that respond to the changes in conditions During Translation:  eIF2 forms a complex with GTP and mediates the binding of the methionyl initiator tRNA to the small ribosomal subunit, which then binds to the 5ʹ end of the mRNA and begins scanning along the mRNA. When an AUG codon is recognized, the eIF2 protein hydrolyzes the bound GTP to GDP, causing a conformational change in the protein and releasing it from the small ribosomal subunit.  The large ribosomal subunit then joins the small one to form a complete ribosome that begins protein synthesis. Regulation of gene expression by eif2  Regulation of gene expression by eif2 Because eIF2 binds very tightly to GDP, a guanine nucleotide exchange factor, designated eIF2B, is required to cause GDP release so that a new GTP molecule can bind and eIF2 can be reused. The reuse of eIF2 is inhibited when it is phosphorylated—the phosphorylated eIF2 binds to eIF2B unusually tightly, inactivating eIF2B. Regulation of gene expression at the post-translation level Post-translation modification  Protein degradation The addition of an ubiquitin group to a protein marks that protein for degradation. Ubiquitin acts like a flag indicating that the protein lifespan is complete. These proteins are moved to the proteasome, an organelle that functions to remove proteins, to be degraded.