229.GeneExp1.110716 - Gene Expression I The Gene0c Code and...

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Unformatted text preview: Gene Expression I: The Gene0c Code and Transcrip0on. 21 11/07/16 Flow of gene0c informa0on: The central dogma of molecular biology. •  The informa2on in DNA is transcribed into (m)RNA •  The mRNA sequence is translated into polypep2des/proteins. •  Mul2ple polypep2des may assemble into func2onal proteins. •  Excep2ons. The gene-­‐protein correla0on: The one gene one enzyme hypothesis. •  Mutagenesis of a yeast strain, generated auxotrophs from prototrophs. •  What did that indicate? •  The mutants were sorted by deficiency for specific metabolite. •  Par2cular enzyme responsible was iden2fied by providing intermediate metabolites. •  Enabled correla2on of each muta2on with “disabled enzyme” and lead to the “one gene on enzyme” hypothesis. Diseases, defec0ve proteins and muta0ons. Ÿ Sickle cell anemia results from defec2ve hemoglobin •  A single amino acid change in hemoglobin alters its physical proper2es and both the structure and func2on of red blood cells. Iden0fica0on of amino acid sequence change in sickle cell hemoglobin. Ÿ Difference between Hbs and Hbn was determined by chromatography. Ÿ  Analysis of tryp2c digests allowed iden2fica2on of altered pep2de. Ÿ Sequencing revealed amino acid change. Triplet nature of the gene0c code-­‐1. •  DNA is made up of 4 nucleo2des and proteins of 20 amino acids. •  Therefore the DNA nucleo2des must combinatorially code for amino acids and the minimum coding segment needs to be comprised of 3 units. Why? •  The 64 combina2ons possible with a triplet code makes for extensive redundancy. Frame shiP muta0ons confirm the triplet code-­‐1. Ÿ Acridine mutagenesis produces single nucleo2de dele2on and inser2on muta2ons. •  These alter the reading frame and lead to a different translated sequence. Frame shiP muta0ons confirm the triplet code-­‐2. Ÿ Certain double muta0ons lead to a pseudo reversion phenotype. •  Only opposite double muta2ons in close proximity lead to reversion if the majority of the reading frame is restored. Frame shiP muta0ons confirm the triplet code-­‐3. Ÿ Triple muta0ons of the same type lead to a pseudo reversion phenotype. Ÿ The gene2c code is non-­‐overlapping and non-­‐ambiguous. Experimental demonstra0on of the gene0c code. •  In-­‐vitro transla2on with cell free extracts and ar2ficial RNAs allowed demonstra2on of the gene2c code. •  Homopolymers of U, A and C revealed the coding proper2es of . •  Subsequently copolymers of 2 nucleo2des allowed provided further evidence of the triplet code and redundancy •  Random copolymer of A and C encodes 8 different codons but produces a polypep2de containing 6 amino acids. •  tRNA binding experiments with ribosomes and ar2ficially synthesized trinucleo2des enabled the assignment of the 64 triplet combina0ons to specific amino acids. •  Synthesis of RNA molecules with defined sequences confirmed the triplet nature of codons. The Gene0c Code. •  The gene2c code is unambiguous and degenerate. •  Some amino acids are encoded by only 1 codon (Met, Trp) others by 2-­‐6 codons. •  What is the advantage of redundancy ? Transcrip0on in prokaryotes-­‐1. •  Prokaryo2c transcrip2on in bacteria is divided into 4 stages-­‐binding, ini0a0on, elonga0on and termina0on •  Binding-­‐RNA polymerase guided by the σ subunit binds promoter regions. •  Ini0a0on:The DNA duplex is unwound and 2 nucleo2des complementary base pair with the template strand and are polymerized by forma2on of phospho-­‐ diester bond. Transcrip0on in prokaryotes-­‐2. •  Elonga0on: When RNA strand is 9n long the σ subunit dissociates from RNA polymerase complex and it goes into elonga2on mode. •  The σ subunit can then re-­‐ ini2ate transcrip2on. •  Termina0on: When the transcrip2on complex reaches the end of the transcrip2on unit termina2on occurs in one of two ways. Promoter sequences define transcrip0on start sites. •  Transcrip2on start sites are defined by specific promoter sequences. •  Two segments at -­‐10 and -­‐35 (rela2ve tss) have been observed in most promoters. •  These sequences are recognized and bound by the σ subunit which then recruits the RNA pol complex. Transcrip0onal Elonga0on. •  In elonga2on theDNA ahead of polymerase unwinds and DNA behind rewinds. Supercoils are resolved by topoisomerase. •  Polymerase also had proofreading ability. It can back up and remove the last (mismatched) nucleo2de and one prior to that. However fidelity of transcrip2on is less stringent than replica2on-­‐why? Transcrip0on termina0on in prokaryotes: ρ dependent. •  Transcrip2on termina2on can be rho (ρ) dependent or independent. •  In ρ dependent transcrip2on when the polymerase nears the end of the unit ρ recognizes and binds sequences 50-­‐90 bp long in the 3’ end of the RNA. •  It then func2ons as an ATP dependent helicase to unwind the RNA and detach if from the DNA. Transcrip0on termina0on in prokaryotes: ρ independent. •  The 3’ end of the unit has a GC rich region followed by a stretch of As. •  Upon transcrip2on GC rich region forms a hair loop structure instead of DNA-­‐RNA helix . •  This is followed by a stretch Us weakens of bonding in this segment allows RNA to detach. Features of eukaryo0c transcrip0on. •  Transcrip2on is carried out by 3 different types of polymerases. •  Eukaryo2c promoters exhibit much more variability and are composed of both core and addi0onal elements. •  A large number of transcrip0on factors (general and specific) facilitate transcrip2on. •  Some transcrip2onal components bind directly to DNA others to proteins. Assembly of transcrip2on complex is sequen2al. •  RNA termina2on mechanisms are also variable. Cleavage of the nascent RNA is crucial for certain types of termina2on. •  Transcrip2on and transla2on are dis2nct and transcripts are extensively processed prior to transla2on. Eukaryo0c RNA polymerases and their a]ributes . •  RNA polymerases are classified according to the sequences/genes they transcribe. •  They differ in protein composi2on and promoter preferences. •  They also differ in their sensi2vity for α-­‐amani0n. Structural organiza0on of eukaryo0c promoters: Pol I. •  These promoters have a core element that flank and define the tss. •  Transcrip2on is boosted by the upstream control element. Structural organiza0on of eukaryo0c promoters: Pol II. •  They have different combina2ons of 4 types of sequence mo0fs. •  Promoters are either TATA driven or DPE (downstream promoter element) driven. These sequences determine the tss. •  BRE s2mulates transcrip2on. Structural organiza0on of eukaryo0c promoters: Pol III. •  Promoter elements for pol III lie wholly downstream of the tss. •  They have two conserved elements that differ in spacing and sequence. Pol II transcrip0on pre-­‐ini0a0on. •  Eukaryo2c transcrip2on involves the stepwise binding of TFs and RNA polymerase. Pol II transcrip0on ini0a0on. •  The complex of TFs and RNA polymerase forms a pre-­‐ ini0a0on complex. •  For Pol II to ini2ate transcrip2on it needs to be released from the pre-­‐ini0a0on complex. •  This is catalyzed by TFIIH which acts as a helicase and also phosphorylates pol II so that it can dissociate from the other TFs. Transcrip0on Elonga0on. •  Elonga0on: Transcrip2on complexes (ini2a2on and elonga2on) include chroma0n remodeling factors. •  They facilitate the disassembly of histones ahead of the polymerase and reassembly behind it. Transcrip0on termina0on. •  Termina0on: is governed by a variety of signals. Pol I: Protein factor recognizes a 18 nucleo0de termina0on signal in the 3’ end of the RNA and dissociates it from DNA. Pol II: Trancrip2on does not have a defined termina2on. Instead the pre mRNA is cleaved at a AAUAAA sequence once it is transcribed and detaches from the DNA, while transcrip2on con2nues. Pol III: The termina2on signal consists of a short stretch of Us which weaken the DNA-­‐RNA duplex and allow dissocia2on without the need for proteins. ...
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