Chapter 9Molecular Mechanisms of

Gene Regulation

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• In prokaryotes, on–off gene activity is often controlled through transcription

• In negative regulation, the default state of transcription is “on” until it is turned “off” by a repressor protein that binds to the DNA upstream from the transcriptional start site

Transcription Regulation: Prokaryotes

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Transcription Regulation: Prokaryotes

• In inducible transcription, a repressor keeps transcription in the “off” state (repressor + inducer on)

• In repressible transcription, the default state is “on” until an active repressor is formed to turn it “off” (aporepressor + co-repressor off)

4Figure 9.1: Negative regulation

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Transcription Regulation: Prokaryotes

• In positive regulation, the default state of transcription is “off” and binding with a transcriptional activator protein is necessary to turn it “on”

• Many systems are both positively and negatively regulated

• Negative regulation is more common in prokaryotes, positive regulation in eukaryotes

• Some genes exhibit autoregulation—the protein product of a gene regulates its own transcription

6Figure 9.2: Transcription

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Operon Model of Regulation

• In bacterial systems, when several enzymes act in sequence in a single metabolic pathway, usually either all or none of the enzymes are produced

• This coordinate regulation results from control of the synthesis of one or more mRNA molecules that are polycistronic

• Polycistronic mRNAs are transcribed from an operon: a collection of adjacent structural genes regulated by an operator

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lac Operon

• The genetic regulatory mechanism in bacteria was first explained by the operon model of François Jacob and Jacques Monod

• They studied lactose-utilization system in E.coli

• The lactose-utilization system consists of two kinds of components: structural genes (lacZ and lacY), which encode proteins needed for the transport and metabolism of lactose, and regulatory elements (the repressor gene lacI, the promoter lacP, and the operator lacO)

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lac Operon

• The products of the lacZ (enzyme -galactosidase) and lacY (transporter lactose permease) genes are coded by a single polycistronic mRNA

• The linked structural genes, together with lacP and lacO, constitute the lac operon

• The promoter mutations (lacP–) eliminate the ability

to synthesize lac mRNA

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lac Operon

• The product of the lacI gene is a repressor, which binds to a unique sequence of DNA bases constituting the operator

• When the repressor is bound to the operator, initiation of transcription of lac mRNA by RNA polymerase is prevented

• Because the repressor is necessary to shut off mRNA synthesis, regulation by the repressor is negative regulation

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lac Operon• Lactose (inducer) stimulate mRNA synthesis by

binding to and inactivating the repressor

Figure 9.4a/b: lac operon

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lac Operon

• In the presence of an inducer, the operator is not bound with the repressor, and the promoter is available for the initiation of mRNA synthesis

Figure 9.4c: lac operon

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lac Operon

• lac operon is also subject to positive regulation

• Positive regulation of the lac operon involves cAMP-CRP (cyclic AMP receptor protein), which binds to the promoter to activate transcription by RNA polymerase

• In the absence of the cAMP–CRP complex, RNA polymerase binds only weakly to the promoter

14Figure 9.6: Four regulatory states of the lac operon

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trp Operon

• The trp operon contains the structural genes that encode enzymes required for the synthesis of the amino acid tryptophan

• The trp operon is transcriptionally active unless tryptophan is present

• The trp operon is an example of a repressible system regulated by a negative feedback loop

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Figure 9.9: The trp operon in E. coli

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trp Operon

• The trp operon is shut off when tryptophan binds to inactive aporepressor

• Tryptophan-repressor complex binds to operator to block transcription when tryptophan levels are high

• If tryptophan levels fall, trp-repressor complex dissociates from operator

18Figure 9.10: Regulation of the E. coli trp operon

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Attenuation

• The trp attenuator consists of 5' base sequence in mRNA that is complementary and can base-pair to form a stem and loop structure

• Attenuation is a very sensitive form of translational regulation of the trp operon

Figure 9.11: The terminal region of the trp attenuator sequence

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Attenuation

• Attenuation results in the premature termination of mRNA synthesis due to stem and loop formation in the 5' region of mRNA

• If tRNATrp is present , synthesis of the leader peptide results in pairing of mRNA, which blocks the action of RNA polymerase

Figure 9.12: The sequence of bases in the trp leader mRNA

21Figure 9.13: Mechanism of attenuation in the E. coli trp operon

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Attenuation

• At low concentrations of tRNATrp, the ribosome stalls, and the mRNA opens so that transcription continues

• Attenuation permits the cell to respond to tryptophan levels by expressing the genes needed for its synthesis when needed

• Attenuation is possible because in bacteria transcription is coupled with translation

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Riboswitches

• Transcription termination can also be triggered by direct binding of a small molecule (vitamin B12 or flavin mononucleotide) to a 5' untranslated leader mRNA

• An RNA leader sequence able to switch between an antiterminator conformation and a terminator conformation is known as a riboswitch

• Comparison of genomic sequences indicates that riboswitches are present in archaea, eubacteria, and eukarya

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Gene Regulation: Eukaryotes

Gene regulation in eukaryotes differs from prokaryotes:

• The processes of transcription and translation are uncoupled

• The primary transcripts of many genes are alternatively spliced to yield different products

• Each gene is regulated by a separate, often multiple promoters

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Gene Regulation: Eukaryotes

Genome organization in eukaryotes:

• DNA in complex with histones forms chromatin

• Significant fraction of DNA is moderately or highly repetitive

• Large fraction of DNA does not code for proteins

• Many coding sequences are interrupted by introns

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Transcriptional Regulation

• Many eukaryotic genes differ in their expression according to cell type or stage of the cell cycle. These genes are often regulated at the level of transcription.

• Galactose metabolism in yeast illustrates transcriptional regulation

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Figure 9.15: Metabolic pathway

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Galactose Metabolism in Yeast

• Despite the tight linkage of the three genes involved in galactose metabolism, they are not part of an operon. The mRNAs are monocistronic.

• The GAL1 and GAL10 mRNAs are synthesized from divergent promoters lying between the genes, and GAL7 mRNA is synthesized from its own promoter.

• These genes are inducible because the mRNAs are synthesized only when galactose is present.

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Figure 9.16: The linked GAL genes of Saccharomyces cerevisiae

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• Constitutive and uninducible mutants have been observed.

• In two types of mutants, gal80 and gal81c, the mutants synthesize GAL1, GAL7, and GAL10 mRNAs constitutively.

• Another type of mutant, gal4, is uninducible: It does not synthesize the mRNAs whether or not galactose is present.

Galactose Metabolism in Yeast

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Table 9.3: Characteristics of Diploids Containing Various Combinations of gal80, gal4, and gal81c Alleles

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Enhancers• Enhancer sequences are typically rather short

(~ 20 bp) and are found at a variety of locations around the gene they regulate

• They may be located many kb from a gene or inside introns and may are able to function as enhancers irrespective of their orientation

• Enhancers are essential components of gene organization in eukaryotes because they enable genes to be transcribed only when proper transcriptional activators are present

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Silencers

• Some genes are also subjected to regulation by transcriptional silencers: short nucleotide sequences that are targets for DNA-binding proteins that promote the assembly of large protein complexes that prevent transcription of the silenced genes

• PcG (Polycomb group) proteins in D. melanogaster silence certain genes during development.

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Chromatin-remodeling Complexes

• Several different multiprotein complexes, known as chromatin-remodeling complexes (CRCs), can restructure chromatin and enable it to be transcribed

• CRCs can disrupt nucleosome structure without displacing them or reposition the nucleosomes making key DNA-binding sites accessible

• CRCs use energy derived from ATP to restructure chromatin

• The molecular mechanism of chromatin remodeling is unknown

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Figure 9.22a/b: Function of chromatin-remodeling complexes

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Figure 9.22c/d: Function of chromatin-remodeling complexes

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Alternative Promoters

• Some eukaryotic genes have multiple promoters that are active in different cell types

• The different promoters result in different primary transcripts that contain the same protein-coding regions

• Alternative promoters make possible the independent regulation of transcription in different tissues or at different stages of development

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Figure 9.23: Use of alternative promoters

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Transcription Activation

• Transcription complex is an aggregate of protein factors that combines with the promoter to initiate transcription

• The basal transcription factors are proteins in the complex that are used in the transcription of many different genes

• The basal transcription factors in eukaryotes have been highly conserved in evolution

• Transcriptional activation occurs by a mechanism called recruitment—the interaction of transcription factors with promoter and enhancers

40Figure 9.20: Transcriptional activation by recruitment

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Transcription Regulation

• Combinatorial control means that a few genes can control many others

• Strategically placed enhancers can act as genetic switches

• One gene can have two or more promoters that are regulated differently

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Epigenetic Regulation

• Epigenetic: heritable changes in gene expression that are due to either chemical modification of the DNA bases, or protein factors bound with the DNA

• In most higher eukaryotes, a proportion of the cytosine base are modified by the addition of a methyl (CH3) group to the number-5 carbon atom

• The cytosines are incorporated unmodified during DNA replication, and then the methyl group is added by an enzyme called DNA methylase

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DNA Methylation

• In mammals, cytosines are modified preferentially in 5'-CG-3' dinucleotides

• Many mammalian genes have CG-rich regions upstream of the coding region that provide multiple sites for methylation—CpG islands

• Heavy methylation is associated with genes for which the rate of transcription is low

• One example is the inactive X chromosome in mammalian cells, which is extensively methylated.

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Imprinting• Mammals feature an unusual type of epigenetic

silencing known as genomic imprinting, a process with the following characteristics: Imprinting occurs in the germ line and affects at

most a few hundred genes It is accompanied by heavy methylation Imprinted genes are differentially methylated in the

female and male germ lines Once imprinted, a silenced gene remains inactive

during embryogenesis Imprints are erased early in germ-line

development, then later reestablished according to sex-specific patterns

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Imprinting

• The epigenetic, sex-specific gene silencing associated with imprinting is dramatically evident in Prader–Willi syndrome and Angelman syndrome characterized by neuromuscular defects, mental retardation, and other abnormalities

46Figure 9.24: Imprinting of genes and varying results

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Alternative Splicing

• Regulation also takes place at the levels of RNA processing

• Alternative splicing is a form of gene regulation that results in the generation of alternative mRNAs from a single gene

• Different splice patterns may occur in different tissues resulting in tissue-specific gene expression

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Alternative Splicing

• Alternative splicing is an important source of human genetic complexity

• Although the number of human genes exceeds

that in worms or flies by a factor of about two, the number of different human proteins may be greater than that in worms or flies by a factor of about five

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Figure 9.25: Alternative splicing of the primary transcript

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RNA Interference

• Different mRNAs can differ in their persistence in the cell

• An important mechanism regulating the stability of RNA transcripts is a phenomenon of RNA interference (RNAi), in which the introduction of a few hundred nucleotide pairs of double-stranded RNA triggers degradation of RNA transcripts containing homologous sequences

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Figure 9.27: Mechanisms of gene silencing by the siRNA and miRNA pathways

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Translation Regulation

• In eukaryotes, gene expression regulated at the level of translation separately from transcription

• The principle types of translational control are: Inability of an mRNA to be translated except under

certain conditions Regulation of the overall rate of protein synthesis Inhibition or activation of translation by small

regulatory RNAs that undergo base pairing with the mRNA

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Figure 9.28: Regulation of translation of target mRNAs

Adapted from S. Altuvia and E. G. H. Wagner, Proc. Natl. Acad. Sci. USA 97 (2000): 9824-9826.

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Translation Regulation

• An RNA sequence complementary to an mRNA is called an antisense RNA

• The antisense regulatory RNAs act by pairing with the mRNA to either inhibit or activate translation

• Regulatory RNAs that control developmental timing in the nematode C. elegans were the first examples of small regulatory RNAs described in eukaryotes.

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DNA Rearrangements

• Some developmental processes are controlled by programmed DNA rearrangements

• A permanent change in DNA sequence implies that the genotype of a cell lineage becomes permanently altered

• Such irreversible changes take place only in somatic cells, so they are not genetically transmitted

• Programmed DNA rearrangement take place in the bone-marrow–derived (B) cells and thymus-derived (T) cells that play key roles in the vertebrate immune system

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Figure 9.30: Formation of a gene for the light chain of an antibody molecule

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DNA Rearrangements

• Programmed transpositions take place in the regulation of yeast mating type

• Many strains of S. cerevisiae have a mating system called homothallism in which some cells undergo a conversion into the opposite mating type

• This allows matings between cells in what would

otherwise be a pure culture

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Figure 9.31: Genetic basis of mating-type interconversion