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. 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. RNA splicing-‐ precisely excision of introns and joining of exons to produce mature mRNAs. The process is mediated by a mulD-‐protein and RNA complex called a spliceosome. Some introns are capable of self-‐splicing, behaving as ribozymes. Introns enable alterna7ve splicing. What purpose does alternaDve splicing serve ? Processing of ribosomal RNAs. 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. 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. TranslaDon occurs at syntheDc factories-‐
ribosomes. • They are ribonucleoprotein complexes that exist as 2 dissociable subunits. Transla7on: Protein synthesis and ribosomes. 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. Transfer RNAs are “charged” by amino acyl tRNA synthetases with their corresponding amino acids . tRNA recognize their cognate codons by base pairing via their an7-‐codons. 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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