CH 16 Prokaryotic Gene Expression Part 1

Prokaryotic Gene Expression Lecture 15 Outline Gene Expression can respond to Environmental Conditions Understand terms and definitions describing regulation Control is often through allosteric interactions Postive and Negative Regulation Cis and Trans Factors Lactose Metabolism in E. coli Partial Diploid Prokaryotic Gene Regulation Most regulation of gene expression in bacteria is transcriptional regulation Certain bacterial genes needed to continuously perform routine tasks undergo constitutive transcription Some genes, needed for responses to changing environmental conditions require regulated transcription Regulation of transcription includes control of both initiation and amount of transcription Gene Regulation Definitions Negative control of transcription involves binding of a repressor protein to a regulatory DNA sequence and preventing transcription of a gene or gene cluster Repressor proteins are a broad category of regulatory proteins that exert negative control of transcription Positive control of transcription involves binding of an activator protein to regulatory DNA and initiating gene transcription Negative Control and Repressors Repressor proteins are a broad category of regulatory proteins that exert negative control of transcription Activated repressors bind to sequences such as those called operators Their binding blocks transcription initiation They can be activated or inactivated via interactions with other compounds Repressor proteins usually have two active sites through which they perform their functions The DNA-binding domain locates and binds operator DNA sequence or other target DNA sequences The allosteric domain binds a molecule or protein, which causes a change in conformation of the DNA-binding domain; this property is called allostery or allosteric interaction Allostery with Repressors Allosteric domains operate in two modes Some repressor proteins undergo inactivation of their DNA-binding domains as a result of allosteric changes brought about by binding an inducer at the allosteric site Other repressors undergo activation of the DNA-binding domain upon allosteric binding to a corepressor Positive Control and Activators Positive control of transcription involves binding of an activator protein to regulatory DNA and initiating gene transcription Positive control of transcription is accomplished by activator proteins that bind regulatory DNA sequences called activator binding sites Activator protein binding facilitates RNA polymerase binding at promoters and helps initiate transcription Activator proteins have a DNA-binding domain Allostery with Activators In one mode of action, the DNA-binding domain is inactive until an allosteric effector compound binds the allosteric domain and induces a conformational change that activates the DNA-binding domain In an alternative mode, certain activator proteins have a DNA-binding domain that is converted to inactive conformation by binding of an inhibitor to the allosteric domain The lac Operon Is an Inducible Operon System under Negative and Positive Control Clusters of genes undergoing coordinated transcriptional regulation by a shared regulatory region are called operons These are common in bacterial genomes; genes in a particular operon nearly always participate in the same metabolic or biosynthetic pathway The lactose (lac) operon of E. coli is responsible for producing three polypeptides needed for use of lactose Glucose produced by lactose breakdown enters glycolysis; the galactose is processed to produce glucose, which then enters glycolysis The breakdown of lactose also produces a small amount of allolactose, which acts as an inducer compound Bacteria with a lac? phenotype are unable to utilize lactose Lac Operon Structure The lac operon consists of a multipart regulatory region and three structural genes The regulatory region contains the promoter that binds RNA polymerase, the operator (lacO) that binds the lac repressor protein, and the CAP-cAMP region These regions partially overlap and are immediately upstream of the start of transcription of the structural genes Lac Operon Structural Genes The three structural genes are lacZ, which encodes b-galactosidase lacY, which encodes the enzyme permease lacA, which encodes transacetylase They are transcribed as a single, polycistronic mRNA, which is translated to produce the three distinct polypeptides LacI The lacI gene is next to, but not part of, the lac operon LacI encodes the lac repressor protein; it is constitutively expressed The lac repressor protein is a homotetramer with a DNA-binding domain that binds to the lacO sequence and an allosteric domain that binds the inducer, allolactose Lac Operon Function The lac operon is transcriptionally silent when no lactose is available or when glucose is available When no b-galactose is produced, there is no allolactose in the cell and the lac repressor protein binds to lacO, preventing transcription This is an example of negative control Lac Operon Function, continued When lactose is available to the cell and glucose is not, transcription of the lac operon is induced With synthesis of b-galactosidase, allolactose is produced, and binds to the allosteric domain of the lac repressor The formation of the inducer-repressor complex alters the DNA-binding domain of the repressor and prevents it binding the operator Additional Control of the lac Operon The inducer-repressor complex alone is not sufficient to generate enough copies of the lac operon mRNA for metabolism of lactose Positive control of the lac operon occurs at the CAP binding site of the lac promoter The site attracts the CAP-cAMP complex composed of the catabolic activator protein (CAP) and cyclic AMP (cAMP) Positive Control of the lac Operon cAMP (cyclic adenosine monophosphate) is synthesized from ATP (adenosine triphosphate) by adenylate cyclase During glycolysis, adenylate cyclase is limited in quantity and little cAMP is produced; therefore when glucose is present in a cell, the CAP-cAMP complex cannot form Without CAP-cAMP bound to the lac promoter region, lac gene transcription is very inefficient; this is called catabolite repression Levels of lac Operon Transcription The lac repressor protein binds reversibly to operator sequences and is occasionally released from lacO Thus there is a very low level of transcription from the operon, even in the absence of lactose A small amount of permease and b-galactosidase proteins allows for import of lactose into a cell and production of allolactose to bind the lac repressor Mutational Analysis Deciphers Genetic Regulation of the lac Operon Genetic analysis of lac operon mutants led to identification of each gene and regulatory region and a functional description of the operon Analysis of Structural Gene Mutations Several dozen lac? mutants were generated by treating E. coli with mutagens Complementation analysis assigned mutations into two complementation groups; these correspond to the lacZ and lacY genes Complementation analysis is carried out in partial diploids produced by conjugation between F? (lac) and F? bacteria Example The genotype of a partial diploid can be written as: F? I? P? O? Z? Y? A? / I? P? O? Z? Y? A? The F? copy of the operon is unable to produce a functional permease (lacY?) and the other copy is unable to produce functional b-galactosidase (lacZ?) However, in combination, the mutations carried by each copy of the operon complement because the wild-type allele of each gene is dominant to the mutant allele Lac Operon Regulatory Mutations Certain mutations of the lac operon lead to constitutive mutants, in which the genes are transcribed continuously whether or not lactose is available Other regulatory mutants cause cells to be unresponsive to the presence of lactose, and therefore lac? Genetic mapping of constitutive mutants localizes them to the lacO and lacI regions Two Components to the lac Operon The discovery of the two sites of constitutive mutations led Jacob and Monod to suggest that the operon functioned under a negative regulatory system They further suggested that both structural genes and regulatory sites were components of the operon Constitutive mutations could affect the production of a regulatory protein (lacI) or its DNA-binding site (lacO) Operator Mutations Lac operator mutations are exclusively cis-acting; they influence transcription of genes only on the same chromosome Normally the lac repressor protein binds operator sequences unless allolactose is bound to its allosteric site Oc mutants have altered operator sequences to which the repressor protein cannot bind and the structural genes are continuously expressed Experiments with Partial Diploids Partial diploids in which an Oc mutation was adjacent to Z? and an O? sequence was adjacent to Z? led to constitutive expression of Z? A second experiment in which the Oc mutation was adjacent to Z? and the O? sequence was adjacent to Z? led to normal expression of Z? Together these two experiments showed that operator is cis-dominant, influencing transcription only of downstream genes Constitutive Repressor Protein Mutations In a haploid cell with genotype lacI?, that is otherwise wild type, the Z and Y genes are constitutively expressed A partial diploid with I? on one chromosome, that also carries Z? and Y?; and I? on the other chromosome, that also carries Z? and Y? has normal expression of both the Z and Y genes This indicates that lacI? produces a regulatory protein that is trans-acting, i.e., is capable of diffusing and interacting with both operators in a partial diploid Nature of lacI? The lacI? gene produces a mutant form of repressor protein that is unable to bind the operator sequence Since the repressor cannot bind the operator, transcription cannot be repressed, leading to constitutive transcription However, in partial diploids, if lacI? is also present, genes will be normally expressed Super-Repressor Protein Mutations A second set of lacI mutations leads to a noninducible operon; the Z and Y genes are not expressed, even in the presence of lactose The mutation causes the allosteric domain to be altered so that allolactose cannot bind to it; the Is (for super-repressor) mutation is dominant to the I? allele This leads to cells that are unresponsive to the presence of lactose Promoter Mutations Mutations of promoter consensus sequences significantly reduce transcription and may eliminate it entirely Most mutations of lacP reduce or eliminate transcription of lacZ and lacY, which are located in cis Active transcription of lac operon genes takes place only when glucose is depleted from the cell and lactose is available to it Summary of Operon Activity Operon Transcription in Absence of Glucose, Presence of Lactose cAMP levels rise due to availability of adenylcyclase CAP-cAMP complex forms and binds the CAP site of the lac promoter Allolactose forms in a side reaction of lactose metabolism Repressor protein binds allolactose, changes conformation, and releases from the operator Molecular Analysis of the lac Operon Molecular analysis has identified the DNA sequences of the lac operon components The repressor protein-binding region overlaps with the promoter-binding location for RNA polymerase, supporting the hypothesis that repressor protein blocks RNA polymerase binding Three distinct segments of operator DNA sequence are identified: O1, O2, and O3 Further Analysis of the Operon Sequences Each of the three segments of the operator sequences interact differently with the repressor protein The repressor protein is a homotetramer with the four polypeptides joined together at their C-terminal ends One end of each bundle forms the operator DNA-binding domain The other end forms the allosteric domain Take Home Message Gene Expression can respond to Environmental Conditions (e.g. utilization of lactose “Lac Operon”) Understand terms and definitions describing regulation (e.g. constitutive expression) Control is often through allosteric interactions (shape changes, e.g. binding of allolactose) Positive and Negative Regulation (e.g. activators and repressors) Cis and Trans Factors (e.g. DNA sequences and proteins that interact with DNA sequences) Lactose Metabolism in E. coli (e.g. LacZ, LacI, Lac0, LacP) Partial Diploid (Complementation Analysis) (Understand if cells can process lactose and what step the would fail, given genotype information) Next Time Next time we will go over Trp Operon in the Prokaryotic Gene Regulation II lecture Homework and reading. Extra Homework problems are available in Sapling. Study Guide to Be released soon.