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. 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. Diﬀerence between Hbs and Hbn was determined by chromatography.
Analysis of tryp2c digests allowed iden2ﬁca2on of altered pep2de. 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. 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. 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. Triple muta0ons of the same type lead to a pseudo reversion phenotype. 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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