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526-301 Biotechnology Protein synthesis in bacteria Dr Mike Dyall-Smith, lab 3.07, [email protected] Aims: • Understand the process of translation in Bacteria • Know the the major components involved • Know the typical genetic signals needed to allow translation of a mRNA • Familiar with the process of protein folding References:, Schaecter et al., Microbe, pp149-58 or Any recent molecular biology textbook. Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Lecture outline: 1. Overview of translation in bacteria (comparison with eucarya) components, genetic code, process. 2. Features of mRNA and tRNA 4. Ribosome structure and function (compared to eucarya) 5. Initiation and termination factors and the process of translation 6. Briefly touch on protein folding. *export and processing covered in future lecture. Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Central dogma DNA Transcription RNA Translation PROTEIN Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 Translation The decoding (translation) of the 3-letter code of nucleotide bases in the mRNA, to direct the synthesis of proteins. A much more complex operation than transcription. 526-301 Biotechnology Overall process of translation in bacteria Initiation factors Requires aa-tRNAs, ATP, GTP Speed: ~ 15 aa/sec mRNA Initiation Elongation Termination Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Protein synthesis in bacteria How do ribosomes bind to mRNA? • Small subunit binds initiation factors • This complex binds to mRNA, initiator tRNA can then bind. • The large subunit joins the complex Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Transfer RNA Acceptor Arm D Arm T Arm Extra Arm Anticodon Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Transfer RNA (tRNA) structure Linkage to aa Arm tRNA Anticodon Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology tRNA is actually ‘L’ shaped Anticodon arm Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology UNIVERSAL GENETIC CODE Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Ribosomes are large enough to bind 2 tRNAs and cover 40 nt of mRNA Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology 70 S Ribosomes Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 Ribosomes contain many proteins and 3 rRNAs 2/3 of the mass of bacterial ribosomes is rRNA Initiation of translation: Initiation of translation: IF-2 puts the fMet-tRNAf initiator in the P site. IF-2, bound to GTP, associates with the P site of the 30S subunit. fMet-tRNAf. then binds to the IF2 on the 30S subunit. IF-2 then transfers the tRNA into the partial P site 50S subunit binds and IF1-3 are released. 526-301 Biotechnology Where do ribosomes bind on mRNA ? Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Ribosome binding sites bind to the 3’ end of 16S rRNA Shine-Dalgarno sequence RBS or SDS Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 Initiator tRNA in bacteria is tRNAf MET N-formyl-methionyl tRNA. It is only used for initiating translation. All proteins start with this amino acid. Internal methionines use another tRNA, tRNAmMET 526-301 Biotechnology tRNA and mRNA move in the same direction through the ribosome Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 A site : site where an aminoacyl-tRNA enters to base pair with a codon. P site : site occupied by a peptidyl-tRNA Deacylated tRNA: has no amino acid or polypeptide chain attached Translocation: the movement of the ribosome, one codon at a time, along mRNA after the addition of an amino acid to the polypeptide chain. Elongation: the stage in a macromolecular synthesis reaction (replication, transcription, or translation) when the nucleotide or polypeptide chain is extended by the addition of individual subunits. 526-301 Biotechnology Two sites for binding tRNAs on the ribosome P A Ribosome movement P and A sites occupied Peptide bond formed Translocation Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Typical polycistronic bacterial mRNA Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Reinitiation on polycistronic mRNA Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 Termination at stop codons: UAA, UGA, UAG Termination codons are recognised by ‘release factor’ proteins (RF1/RF2, RF3). RF1 or RF2 bind to stop codons in the A site of ribosomes, and activate the ribosome to hydrolyse the adjacent peptidyl tRNA (in the P site), to release the protein. RF3 releases the RF1 or RF2. Finally, RRF (ribosome recycling factor) dissociates the remaining mRNA, tRNA and ribosome subunits. 526-301 Biotechnology mRNA degradation in bacteria is rapid Half-life of 1-3 min Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology mRNA degradation in bacteria is rapid Q: What happens if the ribosome reaches the end of a broken mRNA without seeing a stop codon ? A: it gets stalled !! Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology tmRNA - how to overcome stalled ribosomes tmRNA is part tRNA part mRNA. It completes translation, putting a small peptide sequence at the end of a protein. This is a tag for protein degradation. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. http://www.indiana.edu/~tmrna/ Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Overall process of translation in Bacteria Initiation factors Requires aa-tRNAs, ATP, GTP Speed: ~ 15 aa/sec mRNA Initiation Elongation Termination Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Lecture outline: 1. Overview of translation in bacteria (and comparison with eucarya) components, genetic code, process. 2. Features of mRNA and tRNA 3. Ribosome structure and function (compared to eucarya) 4. Initiation and termination factors and the process of translation 5. Outline of protein folding Pictures mainly from Genes V (Lewin) For use by students enrolled in 526-301 Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Signal sequence of HGH This is removed (by the pituitary cells) during secretion, to form the mature hormone. Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Review of Paper: Dr Mike Dyall-Smith, 2007 526-301 Biotechnology ABSTRACT Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Structure of pig growth hormone. alpha helix N C S-S bond 191 aa S.Abdel-Meguid, Monsanto Co. Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Signal sequence of HGH This is removed (by the pituitary cells) during secretion, to form the mature hormone. Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Problems in expressing a eukaryotic protein like HGH in a prokaryote like E.coli • HGH is normally produced as a pre-protein, with a signal sequence (for secretion) that is cut off on export from the cell. • Need to get rid of introns • Need to supply E.coli sequences for transcription and translation. Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Strategy for cloning and expressing HGH • Construct a gene that will express the mature form of HGH (ie. no signal sequence) • Combine cDNA and synthetic DNA to construct the ORF so that the ATG is perfectly positioned with a lac promoter. Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Construction of pHGH107 2x lacUV5 promoters Goeddel et al., Nature 281:544 (1979) HGH and TetR genes transcribed together Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Adjusting the distance between the Shine-Dalgarno sequence and the start codon. In the cloning steps, the spacing was 11bp. This was reduced to 7bp, as in the natural lacZ gene. Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Expression of HGH in E.coli Note the mid-log and stationary phase levels differ. Levels are lower in stationary phase cells. Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Expression of HGH in E.coli pHGH107-1 has the 7bp spacing between SD and ATG (as in lacZ) It is worse than the 11bp version! Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Expression of HGH in E.coli LacI overproducer strain (D1210) shows good control of gene expression (+/- IPTG inducer). Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology HGH production in E.coli - analysed by SDS-PAGE purification from E.coli immuno precipitation pure HGH Multiple bands ?? Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Summary: What they achieved. “This is the first time that a human polypeptide has been directly expressed in E.coli in a non-precursor form.” •Made a gene hybrid (cDNA and synthetic DNA) that coded for the processed form of HGH. •Able to express HGH protein in E.coli at high levels (could purify it easily) •Expressed HGH was able to react with anti-HGH antibodies Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Summary: What they didn’t achieve. • Did it contain f-Met or not? • Was it folded correctly? Did it have the required S-S bridges? • Was it active as a growth hormone? • Why was there so much proteolytic degradation? Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Fig. 1. Construction of plasmid pHGH31, containing the coding sequence (cDNA) for amino acids 24-191 of Human Growth Hormone. Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Plasmid cloning vector cDNA copy of HGH mRNA Cut in the middle of the AmpR gene Tailed cut ends with poly C Tailed 3’ ends with poly G Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Fig 1. E.coli Cloned HGH gene using polyC-polyG ‘sticky ends’ HaeIII and PstI sites regenerated Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Sequence of the Human GH cDNA Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Construction of the sequence coding for aa 1-24 of mature HGH Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Construction of the coding sequence for the first 24aa of mature HGH Small synthetic DNA sequences were assembled and ligated together. The terminal oligos contained restriction site ‘sticky ends’. Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007 526-301 Biotechnology Combining the DNA segments coding for aa 1-24 and aa 24-191 Goeddel et al., Nature 281:544 (1979) Dr Mike Dyall-Smith, 2007