Regulation of Gene Expression
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
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
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.
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
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
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