BIOL 114
Biology Dept
Kenyon College
Chapter 6 B.
Bacterial Gene Regulation
Fall SectionSpring Section 1Spring Section 2

Complementation Analysis                    Lac Operon Quiz -- Highly Recommended
Organization of Genes
Control of Gene Expression
Bacterial Operons
Analysis of Operons
Promoter Structure
Environmental Regulons

Complementation Analysis
Complementation means that two different sources of genetic information (usually, different gene loci encoding proteins or RNAs of different function) together each provide something the other lacks.

Example: Two white-flowered plants cross to produce purple flowers, although purple is dominant.
Each contains a mutation in a different gene, encoding a different enzyme needed to make the purple pigment.

Complementation analysis is easiest to do in bacteria, fungi, or C. elegans, where many mutants of a given phenotype can be obtained.

If we isolate a large number of strains with the same defective phenotype,we can cross them in all combinations, and figure out the number of complementation groups.  Any two defective strains that FAIL to complement are in the same complementation group.  Usually each complementation group represents one of the essential enzymes in the pathway.

Problem 1:  Figure out how many complementation groups there are in these examples.

The concept of complementation is extremely important in molecular biology.  For example, the sickle-cell mouse line could only be created because two strains with different defects (lack of mouse or human  globin genes) could be mated to complement each other's defects.  The fact that genes from different species can complement each other was one of the most significant conceptual advances in molecular biology.  Complementation is now used routinely to answer more subtle questions of how genes are regulated.  You absolutely need to understand complementation to understand molecular biology.

Bacterial complementation.
Bacterial genetic systems can show complementation in two important ways--each manipulating a natural process of bacterial genetics.  These two processes have since been modified in biotechnology to provide most of the essential tools of gene cloning.

1.  Specialized Transduction.  A lysogenic bacteriophage can excise itself so as to carry a piece of host DNA by mistake.  The phage will now carry a second copy of an allele (or linked alleles)  into a host cell.  The new bacterium is a partial diploid for the allele(s).

In biotechnology, a phage chromosome can have a piece of foreign DNA ligated into it in the test tube.  Then the phage DNA is packaged into phage, and it can infect a new host where it either (1) produces many copies of the host gene; or (2) lysogenizes the host, to express the cloned DNA.

2.  F' plasmid.The F plasmid can recombine itself into the host chromosome, then recombine itself out again with some host DNA by mistake.  When it enters the next host cell, it carries a second copy of several genes; again, a partial diploid is created.

In biotechnology, a plasmid can have a piece of foreign DNA ligated into it in the test tube; then the plasmid is transformed into E. coli.  Then the plasmid makes many copies, including the cloned gene.

Suppressor mutation analysis.
A variation on complementation is suppressor mutations.   A suppressor mutation corrects a defect in a different gene locus.  A mutant version of gene A makes an altered gene product, which corrects the phenotype of a defective mutation in gene B.

Organization of Genes
In bacteria, a number of gene ORFs can be organized into an operon.   All the gene sequences in a given operon are transcribed on a single mRNA, starting at one promoter.  An example of an operon is shown, tuf-s10, from Borrelia burgdorferi, which causes Lyme Disease:

This operon encodes ribosomal proteins, and an elongation factor; in all:

  • elongation factor (tuf)
  • ribosomal proteins S10 (rpsJ)
  • L3 (rplC)
  • L4 (rplD)
  • L23 (rplW)
  • L2 (rplB)
  • S19 (rpsS)
  • L22 (rplV)
  • S3 (rpsC)
By contrast, in eukaryotes each gene has its own promoter, often including many kilobases (thousands of base pairs) of regulatory sequences (see below.)   The structure of a eukaryotic gene may be interupted by introns of non-coding sequence.   The segments of coding sequence are called exons.  An example of a human gene sequence is shown:

Human Growth Hormone Receptor

For other interesting operons, try searching GenBank, the international repository for all known DNA sequences.   (Funded by the U.S. government--your tax dollars at work.)

Control of Gene Expression
What controls how much of a gene product is made?
Several levels:

  • DNA sequence--inversion or deletion
  • Transcription, sigma factor
  • Transcription, promoter sequence, repressor proteins
  • Translational repressor
  • Posttranslational modification
In general, DNA sequence change, by "programmed mutation," is the most permanent way of turning a gene on or off, and the least reversible.  For example, the hin recombinase catalyzes the inversion of a DNA segment containing the promoter for a gene encoding flagellin, a protein of the rotary tail of Salmonella.  The recombination event regulates the alternate expression of two flagellin genes, H2 and H1.   These two genes encode proteins with the same function, but different antigenic properties; so, alternate expression helps Salmonella evade your immune system.
The H2 operon is transcribedwhen the promoter is oriented toward  the structural gene H2. This
transcription is followed by translation of the H2 flagellin protein and of the rH1 protein, a repressor of H1 flagellin genetranscription.When the hin recombinase inverts the DNA segment containing the promoter, it turns off H2 transcription and de-represses H1.

Transcription level regulation
Regulation of transcription can respond more quickly, and is more reversible.   Regulation at the level of translation is even more reversible.  In BIOL 14, we will focus on regulation of transcription, or operon control.

The Lac Operon
The Lac operon is the classic model for activation and repression of transcription.  Concepts of analysis based on the Lac operon can be applied to other systems including animals and plants.

The following explanation of the Lac operon is modified from MIT Lac Operon.
Jacob and Monod were the first scientists to elucidate a transcriptionally regulated system. They worked on the lactose metabolism system in E. coli. When the bacterium is in an environment that contains lactose:

 It should turn on the enzymes that are required for lactose degradation. These enzymes are:

This enzyme hydrolyzes the bond between the two sugars, glucose and galactose. It is coded for by the gene LacZ.
Lactose Permease:
This enzyme spans the cell membrane and brings lactose into the cell from the outside environment. The membrane is otherwise essentially impermeable to lactose. It is coded for by the gene LacY.
Thiogalactoside transacetylase:
The function of this enzyme is not known. It is coded for by the gene LacA.
The sequences encoding these enzymes are located sequentially on the E. coli genome. They are preceded by the LacI region which regulates expression of  the lactose metabolic genes.   You might expect that the cell would want to turn these genes on when there is lactose around and off when lactose is absent. But the story is more complicated than that.   For instance, the permease gene always needs to be expressed at a low level, in order for any lactose to get into the cell.  So a certain low level of expression is constitutive--that is, occurs all the time, even if "repressed."  Most bacterial operons are partly or totally constitutive.  LacI expression, for example, is totally constitutive; its promoter is always "turned on," for a very low level of expression, just enough to make a few repressor molecules.

 A bacterium's prime source of food is glucose, since it does not have to be modified to enter the repiratory pathway. So if both glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favour of glucose metabolism. There are regulatory sites upstream of the Lac genes that respond to glucose concentration.

An overall picture of Lac regulation would be this:

  • Lactose induces transcription by pulling the LacI repressor off.
  • Glucose prevents transcription by pulling the CAP activator off.

Element purpose
Operator (o-lac) binding site for repressor
Promoter (p-lac) binding site for RNA polymerase
Repressor (LacI) gene encoding lac repressor protein
Binds to DNA at operator and blocks binding of RNA polymerase at promoter
p-I promoter for LacI
CAP binding site for cAMP/CAP complex

Lac Repressor Tutorial

Catabolite Activator Tutorial

When lactose is present, it acts as an inducer of the operon.  It enters the cell, rearranges slightly to form allolactose, then binds to the Lac repressor.  A conformational change causes the repressor to fall off the DNA. Now the RNA polymerase is free to move along the DNA, and RNA can be made from the three structural genes.  The mRNA will be translated to the proteins which transport and metabolize lactose.

 When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no protein is made.

 Note that RNA polymerase can still bind to the promoter though it is unable to move past it. That means that when the cell is ready to use the operon, RNA polymerase is already there and waiting to begin transcription; the promoter doesn't have to wait for the holoenzyme to bind.

Catabolite Repression, with an Activator Protein
 When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming.  But when glucose levels drop, ATP phosphates are released until at last forming cAMP:

ATP --> ADP + Pi --> AMP + Pi --> cAMP

cAMP binds to a protein called CAP (catabolite activator protein), which is then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for RNA polymerase. This phenomenon is called catabolite repression, a misnomer since it involves an activator protein, but understandable since it seemed that the presence of glucose repressed all the other sugar metabolism operons.

This image shows a "close-up" view of CAP regulation:

Corepressor control
Other operons are controlled by their products, rather than their substrates; for example, expression of biosynthetic enzymes to build amino acids.  This is called feedback inhibition.  In the Trp operon, for tryptophan biosynthesis, transcription of mRNA for five enzymes is prevented by binding of the Trp corepressor in the presence of tryptophan.  When tryptophan levels fall, Trp comes off of the corepressor, and the corepressor comes off of the promoter/operator site.  Transcription now occurs, so that the cell has enzymes to make more tryptophan.

Analysis of operon control
What experiments do we perform to figure out how operons are regulated?
We use partial diploid strains created by F' or specialized transduction.  In either case, we test what happens when a strain is diploid for regulatory elements.

Regulatory mutants can have various kinds of mutant phenotypes.  For example:

p-        Promoter fails to bind RNA polymerase.  No transcription occurs.
lacI-    Repressor fails to bind promoter/operator.  Transcription occurs constitutively
            (in the presence or absence of lactose)
o-c      Operator fails to bind repressor.  Transcription is constitutive.
lacZ-    Structural gene is defective.  No enzyme is made.

What will happen?  What kinds of complementation can occur?Does is matter if the two mutant alleles are adjacent on the same chromosome (cis) or separated (trans)?

"Wild type"
Makes B-gal enzyme
Make NO enzyme
Makes Repressor
Makes NO repressor;
Transcription can be constitutive
p +
RNA Pol binds promoter
p -
RNA Pol does NOT bind promoter;
No Transcription
o +
Operator binds repressor
o - c
Operator does NOT bind repressor;
Transcription can be constitutive

Problem 2.  Predict whether the following diploids produce B-galactosidase, in the presence of lactose; in the absence of lactose.  Explain why.Explain in each case whether it matters if the two mutant alleles are located in cis or in trans.

p +    lacZ -      lacI +
-----------------  ----------
p +    lacZ +      lacI -

p +    lacZ -      lacI -
-----------------  ----------
p -    lacZ +      lacI -

p +    lacZ -      lacI +
-----------------   ----------
p +    lacZ +      lacI -

p +    o-c     lacZ +      lacI +    (A constitutive operator NEVER binds repressor,
--------------------------    --------     with or without lactose.)
p +    o +     lacZ -       lacI +

Problem 3. Explain two different genetic processes in bacteria that can create a "partial diploid" for a small part of the genome.  Explain why these processes are useful for bacterial genetic analysis.

Problem 4. State whether  B-galactosidase is expressed by each lac operon diploid, (1) and (2), and briefly state why (one sentence).  Complete possible genotypes for (3) and (4).

LacI-   P+  O-c  LacZ+
LacI+  P+  O+   LacZ-
LacI+  P-   O-c  LacZ+
LacI+  P+   O+   LacZ-
       LacI-  P+   O+   ___
       ____  ___  __   LacZ- 
        __    P-    O-c   LacZ+
        __     P+   __    ___ 

MIT Bacterial Genetics Problems

Quiz on Lac Operon -- Highly Recommended

Molecular Structure of Promoters
Promoters are defined by sequences of base pairs upstream of the transcription start site.  The RNA polymerase tends to recognize promoter sequences in which most of the base pairs match the promoter consensus sequence.  The consensus sequence is a composite defined by the most common base to occur at each position.  Base-substitution mutations can decrease or increase the efficiency of the promoter.

Bacterial consensus promoters include two regions of six base pairs each, at -10 and -35 bases upstream.  However, no two promoters are exactly alike, and no promoter exactly matches the consensus sequence.  Additional sites for environmental regulators can be found as far as -50 to -300 bases upstream.

Griffiths et al, Genetic Analysis

Environmental Regulons
Genes can be regulated together even though they are located at different parts of the bacterial chromosome.  A group of genes regulated by the same environmental signal is a regulon. An example is the rpoS starvation regulon.

When bacteria enter your digestive tract, how do they adjust to your body's defense mechanisms such as acid and antioxidant stress?  When bacteria exit the intestine how do they cope with starvation?

  • When bacteria use up their carbon sources, they express RpoS, the starvation sigma factor.  (Review, what is a sigma factor?)
  • RpoS joins RNA polymerase to initiate transcription of different environmental stress genes--genes protecting against all the different stresses that the bacteria might encounter before they enter a new human intestine.  This phenomenon is known as cross-protection.
  • The stress genes can be used for conditions as unrelated as acid or base resistance.
  • The stress genes activated may or may not be part of multi-gene operons.  They may face in opposite directions, from many different promoters, at all different loci around the genomic map.
Bacteria are the best genetic system to study genetic regulons.  However, stress genes discovered in bacteria have been shown to have homologs in eukaryotic systems.  For example, heat shock genes have been found in all organisms, including humans.  Heat shock genes in humans also show cross-protection; for example, one heat shock protein interacts with the progesterone receptor and the contraceptive drug RU486.

Research on Operons
  • Arsenic Resistance Operons. How do bacteria resist arsenic?  An environmental response regulon is turned on by arsenic.  The molecular basis is related to  how cancer cells develop resistance to anti-cancer drugs.

An example of an arsenic resistance operon
from Barry Rosen's Arsenic Research Lab.
  • Environmental regulation in Yersinia pestis (bubonic plague bacteria).  Several  complex regulons of genes respond to specific environmental factors, particularly iron and temperature.  At low temperature, the presence of iron tells the bacterium, "I am in blood that has been swallowed by a flea."  The bacterium expresses proteins that upset the flea's digestion, forcing it to regurgitate the bacteria into the blood of its next victim.  (Susan Straley & Robert Perry, Trends in Microbiol., 1995)
  • E. coli virulence regulator.  How do virulent E. coli strains kill children?   A regulator protein binds to an operon encoding "pilins," for E. coli to make pili which attach to the intestinal epithelium.
  • Tuberculosis model gene expression.  How are genes regulated in a tuberculosis-related pathogen?

    M. Donnenberg, U. Maryland
  • Virulence regulator in "Flesh-eating bacteria."  A gene activator protein in Staphylococcus aureus turns on the virulence regulon that makes the flesh-eating toxins.  This activator can be used as a vaccine against S. aureus.

Balaban & Novick, 1995, PNAS 92:1619.
  • DNA Microarrays.  We can now put most of the protein-encoding genes onto a microarray chip, using technology based on the DNA silicon chip industry.  The chip can be used to hybridize to cellular RNA, and measure the expression rates of a large number of genes in a cell.  

Axon Industries.
From "Everything's Great When It Sits on a Chip," The Scientist,  Volume 13, #11, May 24, 1999

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