Lecture 14: Microbial Genetics II

Overview of regulatory mechanisms

- the expression of a gene can be regulated by: 1) controlling the activity of the
enzyme, 2) controlling the amount of enzyme produced (transcriptional control),
3) controlling the transcription of mRNA MOST EFFICIENT
Regulation of gene expression in bacteria
- constitutive enzymes: always present in cell (ex. genes in glycolysis)
- transcriptional control is energy-efficient b/c mRNA and proteins are synthesized
only as they are needed
Negative control of transcription
- repression: decrease amount of mRNA that encodes enzyme v. induction:
increase
- transcriptional regulation: involves allosteric regulatory proteins that bind to DNA
- for negative control repressor protein inhibits mRNA synthesis
- corepressors and inducers are collectively called effectors
- repression (typically affects anabolic enzymes)
o when cells are exposed to a particular end product (corepressor),
synthesis decreases
o ex. synthesis of tryptophan and arginine in E. coli
- induction (typically affects catabolic enzymes)
o in the presence of inducers, cells synthesize more enzymes
o ex. production of B-galactosidase in presence of lactose
Operon model of gene expression
- production of enzymes determined by structural genes
- in bacteria, grp. of coordinately regulated structural genes w/ related metabolic
functions and the promoter and operator sites = operon
- repressible trp operon (tryptophan biosynthesis)
o regulatory gene codes for repressor that is synthesized in an inactive
conformation that wont bind to the operator -> initially genes are
transcribed
o when corepressor (tryptophan) is present, it binds allosteric site of
repressor -> repressor/corepressor complex is active and binds to
operator site and block transcription
o when sufficient amounts of trp are present, biosynthesis is repressed
- inducible lac operon (lactose catabolism)
o regulatory gene codes for active repressor that binds operator and blocks
transcription initially
o when inducer (allolactose) is present, it binds allosteric site of repressor ->
inducer/repressor complex is inactive and cant bind to operator site
o when lactose is present, catabolic enzymes are induced
Global regulatory mechanisms
- global control systems regulate the expression of many genes simultaneously
- catabolite repression is a global control system, helps cells make most efficient
use of carbon surfaces
o for cataboliterepressible enzymes, catabolite activator protein (CAP)
must bind to an activator binding site on DNA before RNA polymerase can
bind
o CAP can bind tot eh DNA activator binding site only if the CAP has first
bound cAMP, an inducer

Lecture 14: Microbial Genetics II

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Lac operon is under the control of catabolite repression + negative regulatory

system
- Catabolite repression in the lac operon
o Transcription require presence of lactose and absence of glucose
o Transcription of structural genes for catabolic enzymes is induced by the
absence of glucose
cAMP acts a cellular alarm (alarmone) that accumulates in absence
of glucose and serves as an inducer for CAP, leading to activation of
transcription
as glucose accumulates, cAMP synthesis shuts down, cell actively
transports cAMP out of cell
in presence of glucose, CAP does not bind and transcription is
repressed
o glucose metabolizing enzymes are produced constitutively + in presence
of preferred substrate, metabolism of alternative carbon sources is
inhibited by catabolic repression
repressors and activators are both allosteric proteins that change
conformation when bound in an allosteric site
Positive control of transcription
- maltose regulon:
o positive control of transcription is implemented by activator proteins
o activator protein activity is modified by effectors
o in presence of effector, effector/activator complex binds to the activatorbinding site on DNA and activates binding of RNA polymerase and
transcription of structural genes
o for positive control of enzyme induction, effector promotes the binding of
activator protein -> stimulates transcription
o ex. of positive control: catabolism of maltose w/ maltose acting as the
inducer (effector) for maltose activator protein
o enzyme for maltose catabolism are encoded by a regulon, multiple
operons under a single regulatory protein
mutations and mutants
mutation: change in the genetic material
- a mutation is a heritable change in the DNA seq. that can lead to a change in the
product coded for by the mutated gene and a change in phenotype
o mutant differs from its parental strain in genotype
- many mutations are neutral (silent), some are disadvantageous (or lethal),
others are beneficial
- types of mutations:
o base substitution (point mutation) occurs when one bp in DNA is replaced
o also: missense mutation (cause AA substitution) or nonsense (early stop
codons)
in missense inactive protein, or one w/ reduced activity, is
encoded
in nonsense incomplete polypeptide is made
o frameshift mutation: one or a few bp are deleted or added -> cause more
dramatic changes in the DNA and result in complete loss of gene function
o spontaneous mutations: occur w/ presence of mutagen

Lecture 14: Microbial Genetics II

frequency of mutation
- mutation rate is prob. that a gene will mutate when a cell divides, expressed as
10 to a negative power (typically 10-7 to 10=11 per bp)
- mutations occur randomly along a chromosome
- RNA genomes typically accumulate mutations at higher frequencies than DNA
genomes
- Low rate of spontaneous mutation is beneficial, provides genetic diversity
Mutagens and mutagenesis
- mutagens are chemical, physical or biological agents in envt that cause
permanent changes in DNA
- chemical mutagens include: bp mutagens, nucleoside analogs, frameshift
mutagens
- ionizing radiation causes formation of ions and free radicals that react w/ DNA ->
cause base substitutions or breakage of sugar-phosphate backbone
- UV radiation is non-ionizing, causing thymine dimers
Repair of mutated DNA
- nucleotide excision repair (aka mismatch repair) can repair by cutting out and
replacing the damaged portion
o only template DNA strand is methylated
o endonuclease recognizes error in nonmethylated strand and makes 2 cuts
to remove short sequence w/ error
o DNA poly. synthesizes complementary replacement strand
o DNA ligase joins replacement strand to original strand
- Photoreactivation enzymes (light-repair enzymes; photolyases) can repair
thymine dimers
- Some DNA damage can lead to cell death if not repaired. Compelx cellular
mechanism called SOS regulatory system is activated as a result of some types
of DNA damage and initiates DNA repair processes, both error-prone and highfidelity (error-free)
Selection and screening of mutants
- selectable mutations: give mutant growth advantage -> if selection is possible,
mutatns must be identified by screening
- identifying mutants: can be detected by selecting for altered phenotype, positive
selection involves rejection of nonmutated cells + selection of mutant cells,
replica plating is used for negative selection (mutants wont grow)
mutagenesis and carcinogenesis: The Ames Test
- Ames test: inexpensive and rapid test for possible chemical carcinogens
- Assumes that mutant cell can revert to a normal cell in presence of mutagen and
that many mutagens are carcinogens
- Reversions to the nonmutant state are selected for