Over 200 different cell types all (almost) have the same have same genome all doing different things e.g., Hb genes expressed only in RBC precursors Differences are due to gene regulation which genes will be transcribed? how transcript will be processed? when mRNA is translated?, how often? Chapter 9

© 2006 Jones and Bartlett Publishers Chapter 9 Molecular Mechanisms of Gene Regulation 9.1Transcription regulation in Prokaryotes 9.2Polycistronic genes - lac operon 9.3Transcriptional termination (attenuation) 9.4Eukaryotic transcriptional regulation 9.5Chemical modifications of the DNA 9.6 Regulation via RNA processing and decay 9.7regulation via translation 9.8programmed DNA rearrangements in development

Regulatory sequences in the DNA Regulatory molecules made by the cell Other Regulatory molecules (may come from cell’s environment)

In bacteria (and bacteriophages) Transcriptional control on:actively transcribed off:very low background levels coordinated regulation synthesis of polycistronic mRNA would this occur in eukaryotes? negative control positive control default is “on” default is “off”

a regulatory molecule binds to a gene preventing transcription repressor binds to operator Negative control

repressor binds to operator region of the gene that binds to the repressor (often composed of inverted repeats) Negative control

© 2006 Jones and Bartlett Publishers Default state of transcription is “on” Repressor binds to operator and turns gene “off” Fig 9.1A

a regulatory molecule binds to a gene facilitating transcription activator binds to activator binding site Positive control

activator binds to activator binding site The region of the gene that binds to the activator Positive control

© 2006 Jones and Bartlett Publishers Fig In positive regulation, the default state of transcription is "off."

a signal from the cell’s environment results in transcription (turns gene “on”) inducer molecule from the cell’s environment that increases transcription Induction

a signal from the cell’s environment inhibits transcription (turns gene “off”) corepressor molecule from the cell’s environment that decreases transcription Repression

Both negative and positive control can involve induction or repression

© 2006 Jones and Bartlett Publishers negative control-induction inducer inactivates the repressor lactose

© 2006 Jones and Bartlett Publishers often found in catabolic pathways Fig 9.1A and B

© 2006 Jones and Bartlett Publishers negative control-repression corepressor activates the repressor (tryptophan)

© 2006 Jones and Bartlett Publishers often found in anabolic pathways Fig 9.1C

© 2006 Jones and Bartlett Publishers positive control-induction inducer activates the activator

© 2006 Jones and Bartlett Publishers positive control-repression corepressor inactivates the activator

© 2006 Jones and Bartlett Publishers Constitutive expression The gene is always transcribed (always “on”)

© 2006 Jones and Bartlett Publishers Chapter 9 Molecular Mechanisms of Gene Regulation 9.1Transcription regulation in Prokaryotes 9.2Polycistronic genes - lac operon 9.3Transcriptional termination (attenuation) 9.4Eukaryotic transcriptional regulation 9.5Chemical modifications of the DNA 9.6 Regulation via RNA processing and decay 9.7regulation via translation 9.8programmed DNA rearrangements in development

metabolism of lactose in E. coli controlled by the lactose operon negative control by induction repsonds to lactose positive control by induction responds to glucose

metabolism of lactose in E. coli controlled by the lactose operon operon group of linked genes sharing promoter and regulatory sequences transcribed as a polycistronic mRNA prokaryotic

metabolism of lactose in E. coli two proteins are required lactose permease get lactose into the cell beta(  -galactosidase enzyme that cleaves lactose into galactose and glucose lacY lacZ

© 2006 Jones and Bartlett Publishers Fig The "on-off" nature of the lac system

inducible transcription of mRNA (its not on but we can turn it on) lactose is the inducer (lactose is what turns it on) no lactose- no permease or  -galactosidase add lactose - quick, transient expression of mRNA both enzymes appear together

© 2006 Jones and Bartlett Publishers Fig. 9.4A,B. The lac Operon model

© 2006 Jones and Bartlett Publishers Fig. 9.4C. The lac Operon model

© 2006 Jones and Bartlett Publishers Fig. 9.4C. The lac Operon model

lac system summary (pp ) i, o, p, z, y, a structural genes encode for proteins z  -galactosidase ypermease regulatory elements repressor i operator o promoter p 1.two kinds of components: structural regulatory

lac system 2.Products are coded by polycistronic mRNA. Linked structural genes, regulatory promoter and operator make up the lac operon (we won’t worry with lacA product now) 3.Promoter mutations (lacP - ) eliminate the ability to synthesize lac mRNA lac system summary (pp )

lac system 4.Product of the lacI gene is a repressor which binds to the operator DNA sequence 5.When the repressor is bound to the operator, initiation of transcription of the lac mRNA is prevented 6.When the inducer is present, it inactivates the repressor, permitting RNA polymerase to bind to the promoter/operator and initiate transcription of the lac mRNA lac system summary (pp )

lac systemlac system summary (pp ) inducible negative regulation positive regulation can be turned on with lactose transcription occurs until turned off by repressor in a few minutes

lac operonadditional observations operator has to be very near promoter (binding of repressor to operator blocks promoter) repressor does not need to be near operator product is a protein that diffuses repressor binding > 1000 x repressor-inducer b-gal > permease > transacetylase

lac operon implies that glucose has an inhibitory effect on lac operon no lac mRNA is made in the presence of glucose glucose affects cAMP levels cAMP made by adenylate cyclase cAMP binds to cAMP receptor protein (CRP) mutation in crp gene or adenylate cyclase prevents transcription of lac mRNA both are needed for lac mRNA

lac operon cAMP-CRP complex must be present for induction of lac operon lacI - mutants or lacO c mutants even with cAMP-CRP binds to promoter region

© 2006 Jones and Bartlett Publishers Fig Structure of cyclic AMP

© 2006 Jones and Bartlett Publishers Fig Four regulatory states of the lac operon

lac operon cAMP-CRP complex must be present for induction of lac operon RNA polymerase only binds strongly to promoter with the cAMP-CRP complex present Two ways to turn off: repressor binding to operator

skip next three slides…..and wait for them to come out as a movie

© 2006 Jones and Bartlett Publishers Fig Base sequence of the control region of the lac operon (left) RNA polymerase binding site repressor binding site CRP-cAMP binding site repressor binding site lacIlacPlacOlacZ

repressor 2 binding sites R RNA Polymerase CRPR

lac operon repression loop repressor protein DNA

lac operon cAMP-CRP complex must be present for induction of lac operon RNA polymerase only binds strongly to promoter with the cAMP-CRP complex present Two ways to turn off: repressor binding to operator or absence of cAMP-CRP complex

lac system inducible negative regulation positive regulation can be turned on with lactose transcription occurs until turned off by repressor only turned on in presence of CRP-cAMP complex (+)

© 2006 Jones and Bartlett Publishers Fig Base sequence of the control region of the lac operon RNA polymerase binding site repressor binding site CRP-cAMP binding site positive regulator no RNA polymerase binding without it glucose present ? no need to use lactose repressor binding site

© 2006 Jones and Bartlett Publishers Table 9.2. Concentration of cyclic AMP in cells growing in media with the indicated carbon sources High cAMPhigh CRP-cAMP complexLow glucose

lac system inducible negative regulation positive regulation can be turned on with lactose transcription occurs, until turned off by repressor (normal) only turned on in presence of CRP-cAMP complex (+) catabolic pathway

lac system summary regulatory elements repressor I lacI operator o lacO promoter p lacP makes repressor which can bind to operator or to lactose (but not both together) site for repressor binding OR site for RNA polymerase binding site for CRP binding

Lac Operon: the Movie

negative control Regulation of Transcription a regulatory molecule binds to the DNA and prevents transcription repressor operator binds to

positive control Regulation of Transcription a regulatory molecule binds to the DNA and facilitates transcription activator activator binding site binds to

induction Regulation of Transcription a signal from the cell’s environment that results in transcription inducer

repression Regulation of Transcription a signal from the cell’s environment that inhibits transcription corepressor

© 2006 Jones and Bartlett Publishers Chapter 9 Molecular Mechanisms of Gene Regulation 9.1Transcription regulation in Prokaryotes 9.2Polycistronic genes - lac operon 9.3Transcriptional termination (attenuation) 9.4Eukaryotic transcriptional regulation 9.5Chemical modifications of the DNA 9.6 Regulation via RNA processing and decay 9.7regulation via translation 9.8programmed DNA rearrangements in development

trp operon genes for the making the amino acid tryptophan negatively regulated by repressor (like lac operon) repressed when tryptophan is present (if you have it, you don’t need to make it) tryptophan is a co-repressor (not an inducer like lactose) operon is repressible (instead of inducible) anabolic pathway

trp operon structural genes for enzymes needed for tryptophan biosynthesis regulatory elements trpE, trpD, trpC, trpB, trpA trp o, trp p, trp a, trp L, trp R

© 2006 Jones and Bartlett Publishers Fig The trp operon in E. coli

© 2006 Jones and Bartlett Publishers Fig Regulation of the E. coli trp operon lots of tryptophan… ….don’t need to make more

trp operon While trp genes are being transcribed… …it starts being translated early in this transcript are two tryptophan codons in high [tryptophan] high [tRNA -trp ] ribosome continues… transcription is terminated

© 2006 Jones and Bartlett Publishers Fig Mechanism of attenuation in the E. coli trp operon

trp operon While trp genes are being transcribed… …it starts being translated early in this transcript are two tryptophan codons in high [tryptophan] high [tRNA -trp ] ribosome continues… transcription is terminated in low [tryptophan] low [tRNA -trp ] ribosome pauses… …transcription continues

© 2006 Jones and Bartlett Publishers Fig Mechanism of attenuation in the E. coli trp operon

© 2006 Jones and Bartlett Publishers Fig Terminal region of the trp attenuator sequence

trp operon attenuation fine-tuning of control of transcription superimposed on the negative control Many operons for anabolic pathways are regulated by attenuators Would this work in eukaryotes?

Riboswitches different configurations of the leader mRNA antiterminator vs. terminator

© 2006 Jones and Bartlett Publishers Fig. 9.14B. Riboswitch [After B.A.M. McDaniel, F. J. Grundy, I. Artsimovitch, and T. M. Henkin Proc. Natl. Acad. Sci. USA 100: © 2003 National Academy of Sciences, U.S.A.] off on terminatorantiterminator

© 2006 Jones and Bartlett Publishers Chapter 9 Molecular Mechanisms of Gene Regulation 9.1Transcription regulation in Prokaryotes 9.2Polycistronic genes - lac operon 9.3Transcriptional termination (attenuation) 9.4Eukaryotic transcriptional regulation 9.5Chemical modifications of the DNA 9.6 Regulation via RNA processing and decay 9.7regulation via translation 9.8programmed DNA rearrangements in development

9.4Eukaryotic gene regulation transcriptional activator proteins enhancers silencers

9.4Eukaryotic gene regulation transcriptional activator proteins binds to upstream DNA essential for transcription of genes that are positively regulated GAL4 fig 9.18

© 2006 Jones and Bartlett Publishers Fig GAL gene regulation in yeast

9.4Eukaryotic gene regulation enhancers short sequences binding sites for transcriptional activator proteins most-upstream from start site

9.4Eukaryotic gene regulation silencers short sequences targets for DNA binding proteins which block transcription

9.4Eukaryotic gene regulation transcriptional complex aggregate (lots of parts) binds to promoter ( transcription) TFIID (includes TATA binding protein; TBP) TAF’s (TBP associated factors) RNA Pol II fig 9.20

© 2006 Jones and Bartlett Publishers Fig Example of transcriptional activation during Drosophila development

© 2006 Jones and Bartlett Publishers Fig Transcriptional activation by recruitment

9.4Eukaryotic gene regulation alternative promoters fig. 9.23

© 2006 Jones and Bartlett Publishers Fig Use of alternative promoters in the gene for alcohol dehydrogenase in Drosophila

© 2006 Jones and Bartlett Publishers Chapter 9 Molecular Mechanisms of Gene Regulation 9.1Transcription regulation in Prokaryotes 9.2Polycistronic genes - lac operon 9.3Transcriptional termination (attenuation) 9.4Eukaryotic transcriptional regulation 9.5Chemical modifications of the DNA 9.6 Regulation via RNA processing and decay 9.7regulation via translation 9.8programmed DNA rearrangements in development

9.5Chemically modify the DNA epigenetic regulation “in addition to” changing the DNA, but not the sequence methylation of cytosine heavy methylationlow transcription

9.5Chemically modify the DNA imprintingmaybe later fig silencing of specific genes

© 2006 Jones and Bartlett Publishers Fig Imprinting genes in chromosomal region 15q11 results in different neuromuscular syndromes

© 2006 Jones and Bartlett Publishers Chapter 9 Molecular Mechanisms of Gene Regulation 9.1Transcription regulation in Prokaryotes 9.2Polycistronic genes - lac operon 9.3Transcriptional termination (attenuation) 9.4Eukaryotic transcriptional regulation 9.5Chemical modifications of the DNA 9.6 Regulation via RNA processing and decay 9.7regulation via translation 9.8programmed DNA rearrangements in development

9.6Eukaryotic gene regulation RNA processing alternative splicing fig. 9.25

© 2006 Jones and Bartlett Publishers Fig Alternative splicing of the primary transcript of the gene encoding the alpha chain of the insulin receptor

9.6Eukaryotic gene regulation RNA persistence short vs. long-lived mRNA

9.6Eukaryotic gene regulation RNAi (interference) ds RNA is made cleaved by dicer protein RISC (RNA-induced silencing complex) binds to/destroys mRNA (transferable) fig. 9.26

© 2006 Jones and Bartlett Publishers Fig Mechanism of RNA interference (RNAi)

© 2006 Jones and Bartlett Publishers Chapter 9 Molecular Mechanisms of Gene Regulation 9.1Transcription regulation in Prokaryotes 9.2Polycistronic genes - lac operon 9.3Transcriptional termination (attenuation) 9.4Eukaryotic transcriptional regulation 9.5Chemical modifications of the DNA 9.6 Regulation via RNA processing and decay 9.7regulation via translation 9.8programmed DNA rearrangements in development

9.7Eukaryotic gene regulation translation small regulatory RNA base pair with mRNA fig antisense RNA

© 2006 Jones and Bartlett Publishers Fig Regulation of translation of target mRNAs by the regulatory RNAs. [After S. Altuvia and E. G.H. Wagner Proc. Natl. Acad. Sci. USA 97: © 2000 National Academy of Sciences, U.S.A.] off on mRNA