1Biochemistry I Fall Term, 2003October 13, 2003Lecture 19: DNA TranscriptionAssigned reading in Campbell: Chapter 10Key Terms:lac Repressor & operatorCAP-cAMP activationRNA polymerase holoenzymeσ subunitRho (ρ-protein)Consensus sequencesClosed & open promoter complexesProcessive polymerizationFidelity of synthesisLinks:(I) Review Quiz on Lecture 19 concepts(I) DNA Transcription Tutorial Flash animation of prokaryotic transcription.(I) Review of some Molecular Genetics Terminology (Lecture handout, Oct. 10, 2003)(S) RNA Polymerase-Promoter Complex: The bacterial enzyme architecture. (Chime)(S) RNA Polymerase Elongation Complex: The yeast enzyme architecture. (Chime, 80K)Note that DNA replication, DNA transcription, and mRNA translation all have these importantfeatures in common:1. Template-directed polymerization of monomer units.2. A requirement for high fidelity (i.e. accuracy) in the information transfer that takes place(DNA --> RNA --> Protein).3. Enzymatic mechanisms that proceed through discrete phases:a) Recognition occurs near the start site on the template.b) Initiation of the chain is the rate-limiting step.c) Elongation of the chain is rapid and processive.d) Termination occurs at specific sites on the template.4. Regulation of initiation frequency is essential.a) Template sequences encode "start" and "stop" information.2b) Accessory proteins act as (on-off) switches and (up-down) modulators.The four additional key terms listed above serve as an organizing principle for this and the nextthree lecture topics.1. The Role of RNA in Protein Synthesis (Gene Expression). A. Enzyme induction in the Eschericia coli lactose operon (lac)1. Definitionsa) constitutive expression occurs at constant rates.b) inducible expression varies with metabolic conditions.c) an operon is the set of genes and control sites that are required for inducibleexpression.2. The organization of the lac operona) lac repressor (lacRep) binds to its operator site (O) turning off transcription of thegene for β-galactosidase (Z).b) lactose, the substrate for β-galactosidase, binds to and inactivates lacRep. Thisallows transcription to start at the promoter site (P), but only at a low level (5 RNAchains /hr).c) If the glucose concentration in the cell is low, cAMP concentration will be high.This causes CAP-cAMP to bind at its activator site (in P), boosting transcription to amaximal level (100 RNAs/hr).3. Analogy to a radioa) lacRep serves as the on-off switch.b) CAP-cAMP functions as the volume control.c) lacZ mRNA is the "music" that comes out of the box.How is the "music" actually made?3 B. Messenger RNA1. cis-acting elements are sites on DNA. In lac, these are the DNA sequences found in Pand O.2. trans-acting factors are proteins. In lac, these are CAP-cAMP and lacRep.3. mRNA is transcribed from the template strand of the Z, Y, and A genes (and the I gene).2. RNA Polymerase A. Enzyme structure1. Core enzyme: α2ββ' catalyzes RNA synthesis from NTP's.2. Holoenzyme (Core + σ): the dissociable σσσσ subunit is essential for recognizing the DNAsequence of the promoter site. B. Template binding1. Holoenzyme (R) binds only to promoter sites (P).2. Promoter consensus sequence (non-template strand)Promoter mutations show that "consensus is best".3. Recognition (binding) is reversible.4. "Open complex" formation.a) an (essentially) irreversible, committed step.b) DNA is melted (from -9 to +2).c) Rate-limiting step in RNA synthesis.4 C. Chain initiation1. The 5' ends of RNA are triphosphorylated.e.g. ATP + NTP --> pppApN + PPi (N = A, C, G, or U)2. When the RNA chain is about 10 nucleotides long, σ subunit dissociates, leaving coreenzyme to elongate the RNA processively (i.e. without dissociating from the DNAtemplate).3. Rifamycins block productive RNA chain initiation.a) Synthesis proceeds only to the dinucleotide (pppApN).b) Rifampicin is an effective anti-tuberculosis drug. But, bacterial resistance torifampicin is becoming rampant, worldwide.D. Chain elongation1. RNA chain growth is from 5' to 3'.2. Elongation is rapid: about 50 nucleotides/sec.3. RNA synthesis occurs with moderate fidelity: about 1 incorrect nucleotide/104incorporated.4. Actinomycin D inhibits elongation by intercalating into the template DNA helix. E. Chain termination1. Termination can occur at specific sites (i.e. DNA sequences).2. Termination usually involves Rho factor.a) Rho protein binds to "naked" RNA.b) The Rho ATPase activity is required for termination.c) Rho probably wraps the RNA around its hexameric structure.DNA Transcription continues on the same outline