New User Special Price Expires in

Let's log you in.

Sign in with Facebook


Don't have a StudySoup account? Create one here!


Create a StudySoup account

Be part of our community, it's free to join!

Sign up with Facebook


Create your account
By creating an account you agree to StudySoup's terms and conditions and privacy policy

Already have a StudySoup account? Login here

Molecular Biology of the Gene

by: Jasmin Roob Sr.

Molecular Biology of the Gene B M B 400

Jasmin Roob Sr.
Penn State
GPA 3.58


Almost Ready


These notes were just uploaded, and will be ready to view shortly.

Purchase these notes here, or revisit this page.

Either way, we'll remind you when they're ready :)

Preview These Notes for FREE

Get a free preview of these Notes, just enter your email below.

Unlock Preview
Unlock Preview

Preview these materials now for free

Why put in your email? Get access to more of this material and other relevant free materials for your school

View Preview

About this Document

Class Notes
25 ?




Popular in Course

Popular in Biochemistry and Molecular Biology

This 0 page Class Notes was uploaded by Jasmin Roob Sr. on Sunday November 1, 2015. The Class Notes belongs to B M B 400 at Pennsylvania State University taught by Staff in Fall. Since its upload, it has received 25 views. For similar materials see /class/233047/b-m-b-400-pennsylvania-state-university in Biochemistry and Molecular Biology at Pennsylvania State University.

Similar to B M B 400 at Penn State

Popular in Biochemistry and Molecular Biology


Reviews for Molecular Biology of the Gene


Report this Material


What is Karma?


Karma is the currency of StudySoup.

You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!

Date Created: 11/01/15
BMB400 Part Four II Chpt 17 Transcriptional regulation by effects on RNA polymerase B M B 400 Part Four Gene Regulation Section II Chapter 17 TRAN SCRIPTIONAL REGULATION EXERTED BY EFFECTS ON RNA POLYMERASE Dr Tracy Nixon made major contributions to this chapter A The multiple steps in initiation and elongation by RNA polymerase are targets for regulation 1 RNA Polymerase has to bind to promoters form an open complex initiate transcription escape from the promoter elongate an terminate transcription See Fig 421 2 Summarizing a lot of work we know that 0 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 quotweakquot spots 3 To learn these facts we need 0 genetic data to identify which macromolecules DNA and proteins interact in a specific regulation event and to determine which base pairs and amino acid residues are needed for that regulation event biochemical data to describe the binding events and chemical reactions that are affected by the specific regulation event Ideally we would determine all forward and reverse rate constants or equilibrium constants which are a function of the ratio of rate constants if rates are inaccessible Although in reality we cannot get either rates or equilibrium constants for many of the steps some of the steps are amenable to investigation and have proved to be quite informative about the mechanisms of regulation mama m mp u z cm 17 mmcnptmnal gunman by men an RNA palymuase 21 REGULATION OF RNAFOLYMERASE 117 h RH Rpclzmzcz RPnE nczmczEczEc K k k gtgt k nngzu39nn clnsa l samplex quotpen unnka plummet fnrma39jnn fnrma39jnn clmrante wrmmaunn 13 m ltlgmmvjnn l r fnld variz nn Cnnunl mag 511ml attenuavms pus actime anutverminams pus sigma m u an m m3 B u mac g ms szBu man can plummet mngpiunn mzlung xan m am 9quot ginngzu39nnramys NM NM BE 51quot vermmaunnfacmxs p Pa clused pmmm camplzx Pa apen pmmnmxcamplzx m z mmal maxmung camplzx use mmalelmga ngcamplzx Ex langa nncmnplex m unman Hangman camplzx BMB4OO Part Four A ll Chpt l7 Transcriptional regulation by effects on RNA polymerase B Methods exist for measuring rate constants and equilibrium constants and newer more accurate methods are now being u ed 1 Classical methods of equilibrium studies and data analysis 0 use low concentrations of enzymes andma e ptions that simplify complex reactions so that they can be treated by definite integrals of chemical ux equatio o manip te an equation into a form that can be plotted as a linear function and derive parameter estimates by slope and intercept values 2 Driven by the success of recombinant DNA and protein purification technology and y the increased computational power in des top computers the classical methods are being replace f y o usmg o amo ts of enzymes to directly include the 39 kinetic studies In this approach th 0 numeric e enzy al integrations of chemical ux equatt us 0 methods based on NonLinear m In mes are used in substrate level quantities 39 ns Kinetic Sim ation east Squares NLLS 0 i 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 confidence Box The equations used in this chapter come from several different sources that use differertnames for the same thing The following li t some of these synonyms Synonymous and related terms KB Kb Keq equilibrium constant for binding KS KB for binding of protein to aspecific DNA sequence KNS KB for binding of protein tononspecific DNA P P2 molar concentration of protein R4 molar concentration of repressor D molar concentration of free DNA DS concentration of free specific DNA DNS concentration of free nonspecific DNA DP molar concentration of DNAprotein complex R4Ds concentration of repressoroperator BMB4OO Part Four 7 II Chpt l7 Transcriptional regulation by effects on RNA polymerase C 39 39 39 to 39 39 binding reactions Several methods are available for measuring the amount of protein that binds specifically to a DNA molecule We have already encountered these as methods for localizing proteinebinding sites on DNA and all are amenable to quantitation Ma39or methods include nitrocellulose lter binding electrophoretic mobility shift 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 depen s on com ining the analysis of more than one experiment to obtain enough information to resolve intrinsic binding free energy from cooperativity energy Fig 422 Protein binding assayed by DNase I footprinting P L iULM39 Incmriligl n ein Concuma oll I 4 V77ii i LT quot m f m r1124 335232 quot I Site 1 I 3 M l39prwi39 Min 139 nu simz M 1 Ii 3 ii In A H q mex I inn a 1 Need touse many orders mm W mm 1 33 of magnitude of P intinn M Data courtesy of Dr Tracy Nixon The most robust technique is DNase I footprinting If you are studying the binding of multiple interacting proteins then it is possible that these proteins are showing cooperativity in their binding to DNA When analyzing such cooperativity by DNase I footprinting the resolution is limited to cooperativities gt05 kca mole and is subject to some critical assumptions Geleshifts also called electrophoretic mobility shift assays or EMSAs are useful when there is no cooperativity or when cooperativity is large relative to site heterogeneity Filter binding studies require knowledge about filter retention efficiencies for the different proteineDNA complexes which can only be empirically determine And always ke p in mind that a 39ng sequences do affect binding affinities and even point mutations can have distant effects In any of these assays we are devising a physical means for measuring a quantity that is related to fractional occupancy BMB400 Part Four 7 II Chpt 17 Transcriptional regulation by effects on RNA polymerase D Measurement of equilibrium constants in macromolecular binding reactions 1 Classical methods with their linear transformation are not as accurate as he onLinear Least Squares NLLS regression analysis but they can serve to show the general approach a The binding constants can be determined by titrating labeled DNA binding sites with increasing amounts of the repressor and measuring amount of protein bound DNA and the amount of free DNA Typical techniques are electrophoretic mobility shift assays or nitrocellulose filter binding Note that for a simple equilibrium ofa single protein binding to a single site on the DNA the equilibrium constant for binding KB is approximated by the 39nVerse of the protein concentration at which the concentration of DNA bound to protein equals the concentration of free DNA Fig 423 Fig 423 Measure KB by EMSA 0 P DP 2 DP DP DP El KB DHP D I When 5 1then 1 K B P If it were possible to reliably determine both the concentration of DNA bound to protein ie DP and the concentration of free DNA D then one could plot e ratio o bound DNA to ree at eac concentration of repressor If the results were linear then the slope of the line would giVe the equilibrium binding constant KB See Fig 424 BMB400 Fig 42 Fig 425 Part Four 7 ll Chpt 17 Transcriptional regulation by effects on RNA polymerase 4 Measure KB from DPD Dp gt Dp lfyou could measureDP and D at each P you could DP measure KB KB lDllPl E1 D 10 When DP 1then I KB 1 1 P P RB slope KB However the error associated with determining Very low concentrations of free or bound DNA is substantial and a more reliable measurement is that of the ratio of bound DNA to total DNA ie DPDm as illustrated in Fig 425 The equation describing this binding curve has a form equivalent to the Michelierenten equation for steadyestate enyzme kinetics Note that the concentration of protein at which half the DNA is bound to protein is the inVerse of KB You can show this for yourself by substituting 05 for DPDm in the equation At this point P lKB Measure KB from DPDtot It is more reliable to measure the fraction of labeled DNA in complex with protein ie DPDtot Substitution of DD01 DP into equation for KB gives DP 2 K P Dltot l KB Pl 10 DP Dltot 10 P 50n BMB400 Part Four II Chpt 17 Transcriptional regulation by effects on RNA polymerase 2 Problems with the classical approach In this classical approach experiments were designed such that more concentrations could be assumed to be unchanging and o observations were manipulated mathematically transformed to a linear equation so that one could plot the transformed data decide where to draw a straight line and use the slope and intercepts to estimate the parameters in question Scatchard plots Lineweaver Burke plots etc Two problems are associated with the older technique 0 Deciding where to draw the straight line is an arbitrary decision for each person doing the analysis and using a linear regression to find the quotbest fitquot line is not justified as two of the assumptions about your data that are needed to justify such a regression are not true 0 There is no accurate estimate of the error in the estimate of the parameter value 3 These limitations have been overcome in the last 5 or so years aided by the advent of recombinant DNA techniques that allow the production of large amounts of the proteins being analyzed and the availability of powerful microcomputers that can carry out the large number of computations required for nonlinear least squares regression analysis NLLS a We can model binding reactions by tabulating the different states that exist in a system associating each state with a fractional probability based on the Boltzmann partition function and the Gibbs free energy for that state AG5 and determine the probability of any observed measurement by the ratio of o the sum of fractional probabilities that give the observation and o the sum of the fractional probabilities of all possible states Where j is the number of ligands bound the fractional probability of a particular state is given by this equation for f3 e AGS RT XP2j ffm 5 As an example consider a one site system such as an operator that binds one protein There are two states the 0 state with no protein bound to the operator and the 1 state with one protein bound Thus one can write the equation for f0 and for f BMB400 Part Four II Chpt 17 Transcriptional regulation by effects on RNA polymerase If we expand the fractional probabilities for each of these fractional occupancy equations we derive equations relating fractional occupancy to a function of Gibbs free energies for binding AG protein concentration P2 and complex stoichiometry j For a single site system we have the following equations 39L Y2 AG RT 6 XP2 1 AG RT 1e Since Gibbs free energy is also related to the equilibrium constant for reactions AG RT ln Keq these free energies can be re cast as equilibrium constants as follows Kb gtlt P2 Y1KbgtltP2 A more complete presentation of this method including a treatment of multiple binding sites can be obtained at the BMB Courses web site httpwwwbmbpsueducoursesdefaulthtm by clicking on BMB400 quotNixon Lecturesquot b Analyzing the data After collecting the binding data we are in a position to analyze the observed data to find out what values for AG or Kb make the function best predict the observations Statisticians have developed Maximum Likelihood Theory to allow using the data to find for each parameter the value that is most likely to be correct For biochemical data the approach that is most appropriate most of the time is global nonlinear least squares NLLS regression Fortunately desktop computers are now powerful enough to do these calculations in a few minutes for one experiment or even for many experiments combined in a global analysis This method has several advantages It gives you BMB400 Part Four II Chpt 17 Transcriptional regulation by effects on RNA polymerase o the same parameter estimates no matter what program or method you or someone else uses provided that the program is written correctly and used correctly 0 much more rigorous estimates of error This last point is worth emphasizing is it not true that 100 minus 50 is much less attractive as a fee for your time than is 100 minus 001 The same can be true for estimates of binding free energies or equilibrium constants 0 Moreover when several experiments are required to estimate a parameter the error in each experiment should be included in the estimate of the parameter Without a global analysis that determines a conglomerate error it is not possible to carefully carry forward the error of one experiment to the analysis of data from additional ones c This analysis produces a plot of the variance of fit or error over a wide range of possible values for the parameter being measured such as the AG for binding The AG value with the smallest error is the most accurate value EMBAUEI PanFuure u chpt 17 anscnpnmal regulaan bye ems unRNA pulymemse Anexample D hs aralysls 15 shuumanxg 42 6 The raw u a39a Shawn thth A 2 2 eftpan l pmdmed the bmmng ewves shewh mnghtpahel nflhat gure These data were thehsuheeteene mnehnearleastrsquares analysis The ems emuahee amt Breach pusslble velue ems are planed thth 42 6 Fur example hue thetthe lewestmahee emth 15 aheuteg 5 hidmule Fig 6 Variance nl Fil vs Free Energy Parameters 2 u 2 Ihh39s nee Enemy dGl AG Gibbs free energny bmdmg In the mstsme Ufa tweme syshem dG AG Gibbs free energny bmdmg In the securd sue mamDst system The ahee nfmfurthe AG fur the cuupemuwty hetweeh pmtems buund at the am sues IS alsn planed These u a39a were kmily pmvlded byDr neeyNtxm BMB400 Hg427 Part Four 7 11 Chpt 17 Tmnscriplional reguiauon by effects on RNA polymerase AG VRT 1n Keq Example of calculating KB from plot of variance of fit vs AG AG 95 kcal mol gives the minimum variance or error AG iRTlnKeq In KB AGFiT 95 kcalmol 161017 059 kcalmol KB 98x106M1 R 198X 10393 kcal deg391 mol391 7 298 K RE 059 kcalmol references for N39LLS Senear and Bolen 1992 Meihods Enzymol 210463 Koblan el al 1992 Meihods Enzymol 210405 Senear el al 1991 J Biol Chem 26613661 Some key BMB400 Part Four II Chpt 17 Transcriptional regulation by effects on RNA polymerase E Insights into the mechanism of lac regulation by measuring binding constants 1 Having gone through both classical and non linear least squares analysis for measuring binding constants let s look at an example of how one uses these measurements to better J J the 39 0 gene quot We know that transcription of the lac operon is increased in the presence of the inducer but how does this occur One could list a number of possibilities each with different predictions about how the inducer may affect the binding constant of repressor for operator KB a Does the inducer change the conformation of the lac repressor so that it now activates transcription This could occur with no effect on K b Does inducer cause the repressor to dissociate from the operator DNA and remain free in solution This predicts a decrease in KB for specific DNA but no binding to nonspecific DNA c Does inducer cause the repressor to dissociate from the operator and redistribute to nonspecific sites on the DNA This predicts a decrease in KB for specific DNA but proposes that most of the repressor is bound to non operator sites Measurement of the equilibrium constants for lac repressor binding to operator and to nonspecific DNA in the absence and presence of the inducer shows that possibility 0 above is correct This section of the chapter explores this result in detail 2 In the absence of inducer the repressor or R4 will bind to speci c sites in this case the operator with high af nity and to nonspecific sites other DNA sequences with lower affinity Fig 428 This is stated quantitatively in the following values for the equilibrium association constant Either equilibrium constant can be abbreviated Keq or KB We will use the term Ks to refer to KB at specific sites and KNS for the KB at nonspecific sites KS 2x1013 M l KNS 2x106M 1 A detailed presentation of some representative data and how to use them to determine these binding constants for the lac repressor is in Appendix A at the end of this chapter This Appendix goes through the classic approach to measuring binding constants 3 The binding constant of lac repressor to its operator changes in the presence of inducer Fig 428 Binding of the inducer to the repressor lowers the affinity of the repressor for the operator 1000 fold but does not affect the affinity of repressor for nonspecific sites For R4 with inducer IltS2x1010M391 KNS2x106M391 BMB400 Part Four 7 II Chpt 17 Transcriptional regulation by effects on RNA polymerase Fig 428 Inducer lowers the KB for repressor binding to operator acrepressor acrepressor nonspecific Site a Operator 3 1 2 g s g KB 2 KS 2 2X1013 M391 KB KNS 2X106 M391 In the presence of inducer acrepressor nonspecific site 88 I2 T g 3 p gt KB KS 2X1010 M391 KB KNS 2X106 M1 4 The difference in affinity for specific Versus nonspecific sites can be described by the specificity parameter which is the ratio between the equilibrium constant for specific binding and the equilibrium constant for nonspecific binding Specificity 107 in absence ofirlducer A 10 in presence of inducer gs l3 gs l3 Note the in the presence of the inducer the specificity with which the lac repressor binds to DNA is decreased 10007fold Even though the repressor still has a higher affinity for specific DNA in the presence of the inducer there are so many nonspeci c sites in the genome that the repressor stays bound to these nonspecific sites rather than in ing the operator ence in the presence of the in ucer the operator is largely unoccupied by repressor and the operon is actively transcribed The regulation of the lac operon Via redistribution of the repressor to nonspecific sites in e genome is coVered in more detail in e next two sections They show the effect of haVing a large number of nonspecific low affinity sites competing with a single high affinity site for a small number of repressor molecules


Buy Material

Are you sure you want to buy this material for

25 Karma

Buy Material

BOOM! Enjoy Your Free Notes!

We've added these Notes to your profile, click here to view them now.


You're already Subscribed!

Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'

Why people love StudySoup

Jim McGreen Ohio University

"Knowing I can count on the Elite Notetaker in my class allows me to focus on what the professor is saying instead of just scribbling notes the whole time and falling behind."

Jennifer McGill UCSF Med School

"Selling my MCAT study guides and notes has been a great source of side revenue while I'm in school. Some months I'm making over $500! Plus, it makes me happy knowing that I'm helping future med students with their MCAT."

Bentley McCaw University of Florida

"I was shooting for a perfect 4.0 GPA this semester. Having StudySoup as a study aid was critical to helping me achieve my goal...and I nailed it!"

Parker Thompson 500 Startups

"It's a great way for students to improve their educational experience and it seemed like a product that everybody wants, so all the people participating are winning."

Become an Elite Notetaker and start selling your notes online!

Refund Policy


All subscriptions to StudySoup are paid in full at the time of subscribing. To change your credit card information or to cancel your subscription, go to "Edit Settings". All credit card information will be available there. If you should decide to cancel your subscription, it will continue to be valid until the next payment period, as all payments for the current period were made in advance. For special circumstances, please email


StudySoup has more than 1 million course-specific study resources to help students study smarter. If you’re having trouble finding what you’re looking for, our customer support team can help you find what you need! Feel free to contact them here:

Recurring Subscriptions: If you have canceled your recurring subscription on the day of renewal and have not downloaded any documents, you may request a refund by submitting an email to

Satisfaction Guarantee: If you’re not satisfied with your subscription, you can contact us for further help. Contact must be made within 3 business days of your subscription purchase and your refund request will be subject for review.

Please Note: Refunds can never be provided more than 30 days after the initial purchase date regardless of your activity on the site.