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Chapter 19 The Regulation of Gene Expression • In bacteria, growth and cell division are controlled by genes. These genes’ expression is controlled by the needs of the cell as it responds to its environment with the goal of increasing in mass and dividing. • Genes that are continuously expressed are constitutive genes or housekeeping genes. Examples include protein synthesis and glucose metabolism. • All genes are regulated at some level, so that as resources decrease the cell can respond with a different molecular strategy. • Prokaryotic genes are often organized into operons that are cotranscribed: In a nutshell, a regulatory protein binds to an operator sequence in the DNA adjacent to the gene array, and controls production of the polycistronic (polygenic) mRNA. • Gene regulation in bacteria and phage seems to be similar in many ways to the gene regulation in eukaryotes, including humans. • Much remains to be discovered; even in E. coli, one of the most closely studied organisms on earth, 35 percent of the genomic ORFs have no attributed function. • (ORFs open reading frame are A stretch of DNA that when translated into an amino acid sequence doesn’t contain an internal stop codon. An ORF can be evidence that a DNA sequence is part of a gene or that codes for a specific portion of the complete protein). • There are two classic examples to understand gene regulation in prokaryotes. The lac operon, an example of inducible operons and the tryptophan (trp)that is an example of repressible operons. Inducible operons, The Lac operon • 1. An inducible operon responds to an inducer substance (e.g., lactose). An inducer is a small molecule that joins with a regulatory protein to control transcription of the operon. • 2. The regulatory event typically occurs at a specific DNA sequence (controlling site) near the protein-coding sequence. Lactose as a Carbon Source for E. coli • E. coli expresses genes for glucose metabolism constitutively, but the genes for metabolizing other sugars are regulated in a “sugar specific” sort of way. When that specific sugar is present, the presence of the sugar stimulates synthesis of the proteins needed to metabolize it. • When the dissacharide lactose is E. coli’s sole carbon source, three genes are expressed: • a. β-galactosidase (bacterial lactase) has two functions: 1. Breaking lactose into glucose and galactose. (Galactose is converted to glucose, and glucose is metabolized by constitutively produced enzymes.) 2. Converting lactose to allolactose (an isomerization). Allolactose is involved in regulation of the lac operon (Figure 19.2). • a. β-galactosidase (bacterial lactase) has two functions: 1. Breaking lactose into glucose and galactose. (Galactose is converted to glucose, and glucose is metabolized by constitutively produced enzymes.) 2. Converting lactose to allolactose (an isomerization). Allolactose is involved in regulation of the lac operon. • b. Lactose permease (M protein) is required for transport of lactose across the cytoplasmic membrane. • c. Transacetylase is poorly understood. The lac operon shows coordinate induction • In glucose medium, E. coli normally has very low levels of the lac gene products. • When lactose is the sole carbon source, levels of the three enzymes increase coordinately (simultaneously) about 1,000-fold. • i. Allolactose is the inducer molecule. • ii. The mRNA for the enzymes has a short half-life. When lactose is gone, lac transcription stops, and enzyme levels drop rapidly. Experimental Evidence for the Regulation of lac Genes • In 1961, Francois Jacob and Jacques Monod proposed the operon model for the control of gene expression in bacteria. • The experiments of Jacob and Monod produced an understanding of arrangement and control of the lac genes. • They worked with mutants strains that allowed them to understand and hypothesized how that regulatory mutations had affected the normal control of gene expression for the operon. Jacob and Monod’s Operon Model for the Regulation of lac Genes • Jacob and Monod’s model of regulation, with more recent information, follows: • An operon is a cluster of genes that are regulated together. The order of the lac genes is shown in Figure 19.4, and Figure 19.5 shows the operon when lactose is absent. • The lacI gene has its own constitutive promoter and terminator, and repressor protein is always present in low concentration. Repressor protein binds the operator (lacO+), and prevents RNA polymerase initiation to transcribe the operon genes (negative control). (Binding of the repressor to the operator is not absolute, and so an occasional transcript is made, resulting in low levels of the structural proteins). • β-galactosidase in wild-type E. coli growing with lactose as the sole carbon source converts lactose into allolactose. The allolactose binds to the repressor and this association produces allosteric shift (changes shapes) and dissociates from the lac operator. Free repressor-allolactose complexes are unable to bind the operator. • By removing the repressor protein from the operator, Allolactose induces expression of the lac operon by allowing transcription to occur. Positive control of this operon • Repressor exerts negative control by preventing transcription. Positive control of this operon also occurs when lactose is E. coli’s sole carbon source, with no glucose present (Figure 19.11). • a. Catabolite activator protein (CAP) binds cyclic AMP (cAMP) (Figure 19.12). • b. CAP-cAMP complex is a positive regulator of the lac operon. It binds the CAP-site, a DNA sequence upstream of the operon’s promoter. • c. Binding of CAP-cAMP complex recruits RNA polymerase to the promoter, leading to transcription. Positive control of this operon • When both glucose and lactose are in the medium, E. coli preferentially uses glucose, due to catabolite repression (glucose effect) • a. Glucose metabolism greatly reduces cAMP levels in the cell. • b. The CAP-cAMP level drops, and is insufficient to maintain high transcription of the lac genes. • c. Even when allolactose has removed the repressor protein from the operator, lac gene transcription is at very low levels without CAPcAMP complex bound to the CAP-site. • d. Experimental evidence supports this model. Adding cAMP to cells restored transcription of the lac operon, even when glucose was present. • Catabolic genes for other sugars are also regulated by catabolite repression. In all cases, a CAP site in their promoters is bound by a CAP-cAMP complex, increasing RNA polymerase binding. The trp operon in E. coli • 1. If amino acids are available in the medium, E. coli will import them rather than make them, and the genes for amino acid biosynthesis are repressed. When amino acids are absent, the genes are expressed and biosynthesis occurs. • 2. Unlike the inducible lac operon, the trp operon is repressible. Generally, anabolic pathways are repressed when the end product is available. Gene Organization of the Tryptophan Biosynthesis Genes • 1. Yanofsky and colleagues characterized the controlling sites and genes of the trp operon. • a. There are five structural genes, trpA through trpE. • b. The promoter and operator are upstream from the trpE gene. • c. Between trpE and the promoter-operator is trpL, the leader region. Within trpL is the attenuator region (att). • d. The trp operon spans about 7kb. The operon produces a polygenic transcript with five structural genes for tryptophan biosynthesis. Regulation of the trp Operon • Two mechanisms regulate expression of the trp operon: a. Repressor–operator interaction. • When tryptophan is present, it will bind to an aporepressor protein (the trpR gene product. The trpR gene is located close to this operon, but it is not part of it). • The active repressor (aporepressor-tryptophan) binds the trp operator, and prevents transcription initiation. Repression reduces transcription of the trp operon about 70-fold. b. Regulation by Transcription termination When there is tryptophan maximal starvation, the expression of the tryptophan operator is maximal, however when there is not starvation, transcription is also controlled by attenuation. Attenuation produces only short (140bp) transcripts that do not encode structural proteins and it can reduce trp operon transcription 8- to 10-fold.. Termination occurs at the attenuator site within the trpL region. The proportion of attenuated transcripts to full-length ones is related to tryptophan levels, with more attenuated transcripts as the tryptophan concentration increases. Together, repression and attenuation regulate trp gene expression over a 560- to 700-fold range.