BMB400 Part Four - II = Chpt. 17. Transcriptional regulation by effects on RNA polymeraseB M B 400Part Four: Gene RegulationSection II = Chapter 17.TRANSCRIPTIONAL REGULATION EXERTED BY EFFECTS ON RNAPOLYMERASE[Dr. Tracy Nixon made major contributions to this chapter.]A. The multiple steps in initiation and elongation by RNA polymerase are targets forregulation.1. RNA Polymerase has to * bind to promoters, * form an open complex, * initiate transcription, * escape from the promoter, * elongate , and * terminate transcription.See Fig. 4.2.1.2. Summarizing a lot of work, we know that: • strong promoters have high KB, high kf, low kr, and high rates of promoter clearance. • weak promoters have low KB, low kf, high kr, and low rates of promoter clearance. • moderate promoters have one or more "weak" spots.3. To learn these facts, we need: • genetic data to identify which macromolecules (DNA and proteins) interact in a specificregulation event, and to determine which base pairs and amino acid residues are needed forthat regulation event. • biochemical data to describe the binding events and chemical reactions that are affectedby the specific regulation event. Ideally, we would determine all forward and reverse rateconstants, or equilibrium constants (which are a function of the ratio of rate constants) ifrates are inaccessible. Although, in reality, we cannot get either rates or equilibriumconstants for many of the steps, some of the steps are amenable to investigation and haveproved to be quite informative about the mechanisms of regulation.BMB400 Part Four - II = Chpt. 17. Transcriptional regulation by effects on RNA polymeraseFig. 4.2.1Pc = closed promoter complex, Po = open promoter complex, ITC = initial transcribingcomplex, IEC = initial elongating complex, EC = elongation complex, ECt = terminatingelongation complex.BMB400 Part Four - II = Chpt. 17. Transcriptional regulation by effects on RNA polymeraseB. Methods exist for measuring rate constants and equilibrium constants, and newer,more accurate methods are now being used.1. Classical methods of equilibrium studies and data analysis o use low concentrations of enzymes and make assumptions that simplify complex reactions so that they can be treated by definite integrals of chemical flux equations o manipulate an equation into a form that can be plotted as a linear function, and derive parameter estimates by slope and intercept values2. Driven by the success of recombinant DNA and protein purification technology, and by the increased computational power in desktop computers, the classical methods are being replaced by o using of large amounts of enzymes to directly include them in kinetic studies. In this approach, the enzymes are used in substrate level quantities. o numerical integrations of chemical flux equations (Kinetic Simulation) o more rigorous methods based on NonLinear, Least Squares (NLLS) regression, and o analyzing data from multiple experiments of different design simultaneously (global NLLS analysis).3. These changes * increase the steps in a reaction that can be examined experimentally * replace the limited set of simple mechanisms that can be analyzed with essentially any mechanism * increase knowledge of error, permitting conclusions to be drawn with more confidenceBox 1: The equations used in this chapter come from several different sources that usedifferent names for the same thing. The following lists some of these synonyms.BMB400 Part Four - II = Chpt. 17. Transcriptional regulation by effects on RNA polymeraseC. Experimental approaches to macromolecular binding reactionsSeveral methods are available for measuring the amount of protein that bindsspecifically to a DNA molecule. We have already encountered these as methods forlocalizing protein-binding sites on DNA, and all are amenable to quantitation.Major methods include nitrocellulose filter binding, electrophoretic mobilityshift assays, and DNase protection assays.Which Experimental Technique is Best? * The kind of observations that can be made about the system differ for different experimental approaches. * These differences lead to specific problems with each technique, * Each technique depends on combining the analysis of more than one experiment to obtain enough information to resolve intrinsic binding free energy from cooperativity energy.Fig. 4.2.2Data courtesy of Dr. Tracy NixonThe most robust technique is DNase I footprinting. If you are studying the bindingof multiple, interacting proteins, then it is possible that these proteins are showingcooperativity in their binding to DNA. When analyzing such cooperativity by DNase Ifootprinting, the resolution is limited to cooperativities >0.5 kcal/mole, and is subject tosome critical assumptions. Gel-shifts (also called electrophoretic mobility shift assays,or EMSAs) are useful when there is no cooperativity, or when cooperativity is largerelative to site heterogeneity. Filter binding studies require knowledge about filterretention efficiencies for the different protein-DNA complexes, which can only beempirically determined. And always keep in mind that flanking sequences do affectbinding affinities, and even point mutations can have distant effects.In any of these assays, we are devising a physical means for measuring a quantitythat is related to fractional occupancy.BMB400 Part Four - II = Chpt. 17. Transcriptional regulation by effects on RNA polymeraseD. Measurement of equilibrium constants in macromolecular binding reactions1. Classical methods with their linear transformation are not as accurate as theNonLinear, Least Squares (NLLS) regression analysis, but they can serve to showthe general approach.a. The binding constants can be determined by titrating labeled DNA binding siteswith increasing amounts of the repressor, and measuring amount of protein-bound DNA and the amount of free DNA. Typical techniques areelectrophoretic mobility shift assays or nitrocellulose filter binding.Note that for a simple equilibrium of a single protein binding to a single site onthe DNA, the equilibrium constant for binding (KB) is approximated by theinverse of the protein concentration at which the concentration of DNA bound toprotein equals the concentration of free DNA (Fig. 4.2.3).Fig. 4.2.3If it