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Gene Expression Overview

8 Pages

Microbiology (Biological Sciences)
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Regulation of Gene Expression OVERVIEW This chapter introduces the principles of metabolic regulation to demonstrate how the various pathways and activities of microorganisms discussed in previous chapters are coordinated. The chapter begins with a discussion of the need for regulation and then describes different mechanisms by which prokaryotes regulate metabolic activities. Allosteric regulation is the fastest because it is at the enzyme, or posttranscriptional, level. Examples of Positive and negative regulation of transcription is discussed in detail. Two examples of translational regulation are presented. The final part of the chapter discusses global regulatory networks where single molecules control expression of multiple genes, using quorum sensing systems as the primary example. OBJECTIVES After reading this chapter you should be able to understand the following: 1) The need for metabolic regulation to maintain cell components at the proper levels to conserve materials and energy 2) Regulation of gene expression by promoters, repressors, corepressors, and inducers. 3) Regulation of gene expression by activators, inducers, and DNA bending to recruit RNA polymerase. 4) Diauxic growth and how it occurs. 5) Combination of positive (CAP-cAMP) and negative (lac repressor) regulation of lac operon and concept of catabolite repression. 6) The importance of global regulation by activators, repressors, and alternative sigma factors for controlling multiple responses to a single environmental signal. 7) Use of antisense RNA and riboswitches to control translation of mRNA. 8) General concept 2-component regulatory systems for controlling gene expression in direct response to environmental signals 9) Use of quorum sensing by microorganisms to coordinate gene expression for biofilm formation, bioluminescence, and establishing microbe-host relationships. CHAPTER OUTLINE I. Prokaryotic Gene Expression A. Organization of genes on DNA strands: A gene is a DNA segment or sequence that codes for a polypeptide, rRNA, or tRNA. In prokaryotes, genes are not usually interrupted by introns as they are in eukaryotes. However, multiple genes can be coded on a single polycistronic mRNA. The open reading frames (ORFs), i.e. regions encoding polypeptides bounded by a start (AUG) and stop codon, are separated by stop codons. Spacer regions, or intercistronic regions, can vary in length from 1-30 nucleotides. Polycistronic mRNAs are generated using single promoter and terminator regions 1 that control transcription by RNA polymerase. The ORFs are located in between the promoter and terminator regions. The promoter is the region of the DNA to which RNA polymerase sigma factor binds in order to initiate transcription. Both DNA strands in the prokaryotic genome can code for RNA, but only one strand is transcribed at any one time. B. RNA Polymerase and Transcription in Bacteria RNA polymerase (a large multi-subunit enzyme) is the enzyme responsible for the synthesis of RNA. The core enzyme of bacterial RNA polymerase is comprised of 4 subunits: α ßß’. Th2 holoenzyme is the core plus a sigma factor. Sigma factors are proteins that associate with RNA polymerase to initiate transcription at specific promoters. Sigma factors bind to the DNA 35 and 10 nucleotides away from the start of transcription. The -10 region is called the “Pribnow box.” The sequences of the -35 and -10 regions of the promoter recruit different sigma factors, which are expressed under certain environmental conditions. This is one way that bacteria regulate gene expression – through the activities of sigma factors (e.g. sporulation is governed by a cascade of sigma factor activity that induce expression of certain sporulation genes at given times during spore formation). The sigma factor dissociates from the core RNA polymerase enzyme once transcription is fully engaged. Terminators are regions of the DNA that, when transcribed, result in the termination of the transcription process. A frequent terminator sequence is UUUUUU since this sequence isn’t strong enough to keep the RNA/DNA hybrid together. A near-by stem-loop structure makes RNA polymerase pause at the UUUUUU sequence so unwinding can occur. Aside from polycistronic mRNA, another major difference between prokaryotes and eukaryotes is the coupling of transcription and translation, which leads to rapid gene expression and regulatory events. Because of the manner in which mRNA is transcribed by eukaryotes by capping and polyadenylating each specific mRNA before it is released to the endoplasmic reticulum, eukaryotes do not have this flexible and rapid manner of translating multiple peptides at once. In other words, for eukaryotes, one mRNA = one peptide translated following transcription and modification; for prokaryotes, one mRNA = multiple peptides translated immediately following transcription. Prokaryotes have polysomes which are mRNA strands covered with multiple ribosomes that simultaneously translate the sequence into polypeptides. II. Regulation of Gene Expression Metabolic regulation is necessary to maintain cell components at appropriate levels and to conserve materials and energy. If a substrate were not available, producing enzymes required for its catalysis would be wasteful and would divert raw material and energy that could be better used for necessary activities. If a particular end product were already abundant in the cells, it would be wasteful to continue producing enzymes that manufacture more of that end product. Mechanisms of metabolic regulation 1. Metabolic regulation of enzyme activity: posttranslational modification of enzymes effects 2 the most rapid change in their activity. The example examined in previous chapters for posttranslational regulation is allosteric control of enzymes in amino acid biosynthetic pathways. 2. Regulation at transcriptional level – prevent formation of mRNA 3. Regulation at translational level – prevent translation of mRNA into polypeptides III. Regulation of mRNA Synthesis – transcriptional control Regulation at the level of mRNA synthesis provides a long-term regulatory mechanism that can respond to major changes in environmental conditions. This type of regulation is even more conservative of materials and energy than posttranslational processes, because the energy for enzyme synthesis does not have to be expended. Consequently, the response to changing conditions is not as rapid as posttranslational control. Sigma Factors and Promoters Let us review transcription of DNA by the RNA polymerase. RNA polymerase of E. coli is about 480,000 Dalton. It is comprised of four types of polypeptide chains. The sigma (σ) chain has no catalytic activity, unlike the other chains. Its major function is in contacting specific regions of the DNA called the promoter to direct where the gene is to be transcribed by RNA polymerase. The site of DNA binding and transcription initiation is in the ß subunits of the RNA polymerase. The start point of transcription is at a T or C base in the DNA. Thus, a A or G base starts the sequence for mRNA. The promoter sequence has two important regions: a short AT-rich region about 10 base pairs upstream of the transcription start site (-10 sequence), and a region about 35 base pairs upstream (-35 sequence). RNA polymerase binds to both of of these sites. L. Synder and W. Champness. 1997. Molecular Genetics of Bacteria. ASM Press, Washington DC, p. 48 The illustration below shows the RNA polymerase moving along the two DNA strands (panel A) to generate complementary mRNA in the 5’ to 3’ direction. The sigma factor and a purine nucleotide (PuNTP) are joined to RNA polymerase, as it reaches the promoter binding sites. The 3 two strands of DNA are unwound and the RNA polymerase begins transcription of mRNA at the 0 locus (panel B). The +1 locus represents the first copying of the DNA strand by a complementary purine base (A or G). Once transcription begins, the sigma factor dissociates from the complex (panel C). The promoter sequence is thus not transcribed. The RNA L. Synder and W. Champness. 1997. Molecular Genetics of Bacteria. ASM Press, Washington DC, p. 49 polymerase continues along the “DNA bubble” adding base pairs and lengthening the mRNA (panel C). Finally, the RNA polymerase encounters a terminator sequence. The polymerase is released along with a copy of mRNA, and the segment of DNA returns to its normal structure. 4 The Operon In bacteria, the genes that encode the enzymes of a metabolic pathway are arranged in a consecutive manner to form a functional unit called an operon. The operon consists not only of structural genes (those that encode enzymes for metabolism) but also regulatory genes (those that initiate or block transcription). Different sigma factors recognize different sets of promoters. The most common are σ 70 promoters, or the “housekeeping” promoters. The superscript stands for the molecular weight of the sigma factor in kDa that recognizes the promoter sequences. Flagella and chemotactic proteins are produced by σ factors. Substitution of the sigma factors immediately changes gene expression because they recognize different -10 and -35 regions of promoters. Negative control of the operator and promoter: enzyme repression and enzyme induction The two varieties of negative control both involve a repressor protein that binds to the operator region, downstream from the promoter. When bound to the operator, the repressor protein overlaps the promoter region (adjacent to the operator), and thereby prevents RNA polymerase from attaching to the promoter, stopping transcr
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