229.GeneExp2.110916 - Gene Expression II RNA processing...

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Unformatted text preview: Gene Expression II: RNA processing, transla7on and protein sor7ng. Chapter 21 and 22 11/09/16 Post transcrip7onal processing of eukaryo7c mRNAs. •  Precursors of mRNAs (pre-­‐ mRNAs) undergo 3 major types of processing •  At the 5’end of the mRNA acquires a methyl guanidine cap. •  This both protects the mRNA from exonucleases and also helps in ribosome recogni7on . •  The AAUAAA sequence near the 3’ terminus of the mRNA acts as a signal for cleavage of the pre-­‐mRNA and polyadenyla7on. •  The polyA tail provides stability to the mRNA. •  It also facilitates export of the mRNA. Poly-­‐adenyla7on of mRNAs. Discovery of introns. Ÿ In most eukaryoDc pre-­‐mRNAs, sequences encoding for amino acids (exons) and interrupted by intervening sequences (introns) •  The existence of intronic sequences in genes relaDve to mRNAs were demonstrated by R loop experiments. Removal of introns: RNA splicing. Ÿ  RNA splicing-­‐ precisely excision of introns and joining of exons to produce mature mRNAs. Ÿ  The process is mediated by a mulD-­‐protein and RNA complex called a spliceosome. Ÿ Some introns are capable of self-­‐splicing, behaving as ribozymes. Introns enable alterna7ve splicing. What purpose does alternaDve splicing serve ? Processing of ribosomal RNAs. Ÿ The 3 major rRNAs are produced by cleavage from a single precursor transcript. •  MulDple/ tandem copies of the pre-­‐rRNA genes are present mulDple chromosomes. Why ? Processing of transfer RNAs. Ÿ tRNAs (transfer RNAs) are synthesized from individual genes and then processed to produce the mature molecule. •  Both rRNA and tRNA molecules undego modificaDons of mulDple residues. These contribute to stability. Transla7on: Protein synthesis and ribosomes. Ÿ TranslaDon occurs at syntheDc factories-­‐ ribosomes. •  They are ribonucleoprotein complexes that exist as 2 dissociable subunits. Transla7on: Protein synthesis and ribosomes. Ÿ The subunits come together on mRNA molecules to catalyze protein synthesis. •  The ribosome has 3 sites-­‐A (amino acyl); P (pep7dyl) and E (exit) site where different tRNAs bind or exit the ribosome. Transfer RNAs bring amino acids to ribosomes and mRNA. Ÿ  Transfer RNAs are “charged” by amino acyl tRNA synthetases with their corresponding amino acids . Ÿ  tRNA recognize their cognate codons by base pairing via their an7-­‐codons. Ÿ  How many different tRNAs are used for translaDon? Wobble base pairing between codons and an7-­‐codons. •  OUen a single tRNA can recognize and base pair with mul7ple codons. •  This occurs due to wobble base pairing between the 3rd nucleo7de of the codon and the corresponding nucleoDde in the anD-­‐codon. How ? UnconvenDonal base pairing UnconvenDonal base usage Steps in transla7on: Ini7a7on. •  The small ribosomal subunit, the iniDator tRNA and iniDaDon factors assemble at the 5’ end of the mRNA. •  This complex translocates on the mRNA to reach the first AUG. •  Then the large ribosomal subunit joins the complex and the iniDator tRNA occupies P posiDon. Steps in transla7on: Elonga7on. •  Then the next charged tRNA comes into the A site which is posiDoned over the 2nd codon. •  Pep7dyl transferase acDvity of the ribosome synthesizes pepDde bond between the amino acids and the ini7ator tRNA gets uncharged. Steps in transla7on: Elonga7on. •  Then the ribosome shiUs on mRNA by 3 nucloDdes and the iniDator codon occupies E site carrying the discharged tRNA which is released. •  The process is repeated and a new charged tRNA comes to A site. Steps in transla7on: Termina7on. •  TerminaDon when the ribosome (A site) reaches a nonsense codon. •  A tRNA release factor binds to the A site. •  The poly-­‐pepDde is cleaved from the tRNA and released. •  The ribosomal subunits dissociate and are recycled. •  Growing and nascent protein associate chaperones that enable proper folding. Protein sor7ng : Cotransla7onal import-­‐1. Proteins that need to be trafficked to the ER, Golgi or out of the cell are co-­‐ translaDonally trafficked through the ER membrane. Protein sor7ng : Cotransla7onal import-­‐2. Proteins that need to be trafficked to the ER, Golgi or out of the cell are co-­‐ translaDonally trafficked through the ER membrane. Protein sor7ng: Post-­‐transla7onal import-­‐1. •  Translated protein is and prevented from misfolding by binding to chaperones. •  The N terminal transit sequence binds to the receptor translocase and is translocated across the membrane(s). •  The transit sequence is cleaved by pepDdase as it emerges. Protein sor7ng: Post-­‐transla7onal import-­‐2. •  In the lumen/matrix the protein is folded properly by Hsp 60 chaperone proteins. ...
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