Chapter 12 Molecular Mechanisms of Gene Regulation 第十二章 基因调控的分子机理

Chapter 12 Molecular mechanisms of gene regulation 12.1 Transcriptional regulation in prokaryotes 12.2 Lactose metabolism and the operon system of gene regulation 12.3 Regulation of the tryptophan operon 12.4 Regulation in bacteriophage lambda 12.5 Transcriptional regulation in eukaryotes 12.6 Epigenetic mechanisms of transcriptional regulation 12.7 Regulation through RNA processing and decay 12.8 Translational control 12.9 Programmed DNA rearrangements

Gene Regulation Gene regulation: The control of synthesis of particular gene products. Gene regulation: The process by which gene expression is controlled in response to external or internal signals. Gene product: Polypeptide or RNA molecule.

Control points for gene expression include:

Transcriptional regulation in prokaryotes Coordinate regulation: Control of synthesis of several proteins by a single regulatory element; in prokaryotes, the proteins are usually translated from a single mRNA molecule. The coordinate regulation results from control of the synthesis of one or more polycistronic mRNA molecules (in contrast, eukaryotic mRNA is monocistronic) encoding all of the gene products that function in the same metabolic pathway.

The molecular mechanisms of regulation The molecular mechanisms of regulation ususlly fall into either of two broad categories: negative regulation and positive regulation.

Negative regulation (Inducible and repressible systems of) negative regulation: Regulation of gene expression in which mRNA is not synthesized until a repressor is removed from the DNA of the gene. In a system subject to negative regulation, the default state is “on”, and transcription takes place until it is turned “off” by a repressor protein that binds to the DNA upstream from transcriptional start site. A negatively regulated system may be either inducible or repressible, depending on how the active is formed.

Inducible systems of negative regulation: In inducible transcription, a repressor DNA-binding protein normally keeps transcription in the “off” state. In the presence of a small molecule called the inducer, the repressor binds preferentially with the inducer and loses its DNA-binding capability, allowing transcription to occur. Many degradative (catabolic) pathways are inducible and use the initial substrate of the degradative pathway as the inducer.

repressible systems of negative regulation In repressible transtription, the default state is “on” until an active repressor is formed to turn it “off”. In this case the regulatory protein is called an aporepressor, and it has no DNA-binding activity on its own. The active repressor is formed by the combination of the aporepressor and a small molecule known as the co-repressor. Presence of the co-repressor thereby results in the cessation of transcription. Repressible regulation is often found in the control of the synthesis of enzymes that participate in biosynthetic (anabolic) patheways; in these cases the final product of the pathway is frequently the co-repressor.

Negative regulation

Positive regulation Positive regulation: Mechanism of gene regulation in which an element must be bound to DNA in an active form to allow transcription. In contrast to negative regulation, a positively regulated system in which the default state is “off”, and binding with a regulatory protein is required to turn it “on”. Such a regulatory protein is called a transcriptional activator protein. Some genes exhibit autoregulation, which means that the protein product of a gene regulates its own transcrition.

Positive regulation

Monod and Jacob proposed the operon theory of gene regulation

operon Operon: A collection of adjacent structural genes regulated by an operator and a repressor. Operator: A regulatory region in DNA that interacts with a specific repressor protein in controlling the transcription of adjacent structural genes. Repressor: A protein that binds specifically to a regulator sequence adjacent to a gene and blocks transtription of the gene.

Regulation of the lactose operon

Lactose utilization

The genes and regulatory units involved in the control of lactose metabolism

The structural genes of the lac operon

Lac- mutants

Mutational and structural analysis of repressor

Inducible and constitutive synthesis and repression

The “on-off” nature of the lac system

The PajaMo experiment

The operon model

Table 12.1 Characteristics of partial diploids containing several combinations of lacI, lacO and lac P alleles

Various genotypes of partial diploids

Constitutive mutation Constitutive mutation: A mutation that causes synthesis of a particular mRNA molecule (and the protein that it encodes) to take place at a constant rate, independent of the presence or absence of any inducer or repressor molecule.

I- and Oc mutations

Is mutation

The operon system of transcriptional regulation

The operon model

Positive regulation of the lactose operon

Catabolite repression

Four regulatory states of the lac operon

How lactose and glucose function together to regulate transcription of the lac operon?

How regulatory proteins interact with RNA polymerase?

The regulatory region of the lac operon

Structure of the lac operon repression loop

Lac repressor protein binds to the DNA

Regulation of the tryptophan operon

The E. coli trp operon

Organization of controlling sites and the structural genes of the E Organization of controlling sites and the structural genes of the E. coli trp operon

Regulation of the E. coli trp operon

Tryptophan acts as a corepressor

Attenuation Attenuation: The attenuation mechanism controls whether transcription, once started, will continue through the operon or be terminated prematurely. Attenuator: A regulatory base sequence near the beginning of an mRNA molecule at which transcription can be terminated; when an attenuator is present, it precedes the coding sequences.

The terminal region of the trp attenuator sequence

The sequence of nucleotides in the trp leader mRNA

The sequence of the leader region of the trp mRNA

Model of the attenuation

Attenuation in the trp operon of the E. coli

Attenuation in the trp operon

Structure of TRAP protein bound to trp RNA

Leader peptide and nucleotide sequence of the histidine operon

Regulation in Bacteriophage Lambda

Bacteriophage λ infects E Bacteriophage λ infects E. coli, each infected cell can undergo one of two possible outcomes

Plaques formed by bacteriophage λ

Mutants of regulatory genes in λ

Map of phage λ

Genetic map of λ bacteriophage

Genetic and transcriptional map of the control region of bacteriophage λ

Expression of Bacteriophage λ

Repression of the lambda lytic genes in lysogenic E. coli cell

Repression of the lambda lytic genes

Genes and recognition sites involved in the lambda lytic regulatory cascade

Functions of the cro gene product in lambda lytic growth

Gene regulation in eukaryotes

Gene regulation is more complex in eukaryotes than in prokaryotes

Key regulatory differences between eukaryotes and prokaryyotes

Transcriptional regulation in eukaryotes

How cis-acting and trans-acting elements influence transcriptions?

Three RNA polymerases

Promoters Promoters, which have counterparts in bacteria, consist of cis-acting nucleotide sequences that serve as the recognition point for RNA polymerase binding. Therefore, they represent the region necessary to initiate transcription and are located immediately adjacent to the genes they regulate. Promoter: A DNA sequence at which RNA polymerase binds and initiates transcription.

Promoter

Enhancers and Silencers Enhancer: A base sequence in eukaryotes and eukaryotic viruses that increases the rate of transcription of nearby genes; the defining characteristics are that it need not be adjacent to the transcribed gene and that the enhancing activity is independent of orientation with respect to the gene. Enhancer is additional cis-acting DNA sequences. Silencer: A nucleotide sequence that binds with certain proteins whose presence prevents gene expression.

Characteristics of enhancers

Enhancers

Structure of the enhancer of the SV40

Basal transcription factors

Transcriptional activators

Repressor proteins

Activator proteins function in cell as dimers

Structural motifs within different types of transcription factors

Helix-turn-helix or homeodomain

Zinc finger

Leucine zipper

Assembly of the transcription complex in eukaryotes

The influence of multiple transcription factors

Transcriptional activation by recruitment

An example of transcriptional activation

Galactose metabolism in yeast

Galactose metabolism in yeast

The characteristics of mutants for galactose metabolism

The CAL1/GAL7/GAL10 genes system

The GAL1 and GAL10 genes in yeast

Model for the activation of the GAL genes of yeast

GAL4 protein bound to DNA

DNA-binding domain of the GAL4

Chromatin-remodeling complexes Chromatin remodeling: Eukaryotic DNA is typically found in the form of chromatin packaged with nucleosomes.Therefore, the normal structure of chromatin is thought to be sufficient to significantly repress gene activity. As a result, activation of genes requires what is called chromatin remodeling, whereby the conformation of chromatin is altered in a way that the proteins of the nucleosome are released from the DNA, allowing it to become accessible to transcription factors and RNA polymerase. Chromatin-remodeling complexes (CRCs): Any of a number of complex protein aggregates that reorganizers the nucleosomes of chromatin in preparation for transcription.

Chromatin structure plays a critical role in eukaryotic gene regulation

Function of chromatin-remodeling complexes

Alternative promoters

Regulation by competition for an enhancer

12.6 Epigenetic mechanisms of transcriptional regulation Epigenetic: Inherited changes in gene expression resulting from altered chromatin structure or DNA modification (usually methylation) rather than changes in DNA sequence. 1. Cytosine methylation 2. Methylation and transcriptional inactivation 3. Cosuppression through transcriptional silencing 4. Genomic imprinting in the female and male germ lines

Cytosine Methylation

Detection of methylated cytosines

Cosuppression through transcriptional silencing Transcriptional cosuppression: A mechanism of gene silencing in which the silenced genes fail to be transcribed. Transcriptional cosuppression: This is a phenomenon in which extra copies of a gene result in the transcriptional silencing-not only of the extra copies but of all copies present in the genome. Transcriptional cosuppression was originally discovered in attempts to genetically engineer plants by introducing extra copies of genes into the genome.

Transcriptional cosuppression: an example

Heterochromatin formation can lead to transcriptional silencing

Genomic imprinting Genomic imprinting: Expression of certain genes is determined by whether the gene is inherited from the female or male parent, a phenomenon called genomic imprinting or parental imprinting. In other words, one or the other parent makes the offspring genetically, leading to functional differences between homologous alleles. Genomic imprinting: A process of DNA modification in gametogenesis that affects gene expression in the zygote; one probable mechansim is the methylation of certain bases in the DNA.

Characteristics of genomic imprinting

Genomic imprinting

Genomic imprinting at the lgf2 locus in the mouse

Imprinting of genes in chromosomal region 15q11

12.7 Regulation through RNA processing and decay 1.Alternative splicing 2. Messenger RNA stability 3. Cosuppression through RNA turnover 4. RNA interference (RNAi)

Alternative splicing

Alternative splicing

Alternative polyadenylation and alternative splicing

Alternative splicing

Alternate splicing

mRNA editing

Messenger RNA stability A short-lived mRNA produces fewer protein molecules than a long-lived mRNA, so features that affect the rate of mRNA stability affect the level of gene expression. Two route of degradation are the deadenylation-dependent pathway and the deadenylation independent pathway.

Cosuppresion through RNA turnover Transcriptional cosuppresion is a mechanism in which the presence of a transgene introduced by genetic engineering results in methylation-associated transcriptional silencing of both the transgene and endogenous copies of the gene. This type of cosuppression is mediated by homology between the promoter sequences of the silenced genes. Posttranscriptional cosuppresion is a mechanism of gene silencing in which the genes are actively transcribed, but no RNA accumulates because the transcripts are rapidly degraded. It is mediated by homology between the transcripts of the silenced genes, and it is highly sensitive to promoter strength and gene dosage, which suggests that the silencing is triggered by the accumulation of transcripts above some threshold level.

RNA Interference RNA Interference is a phenomenon probably related to posttranscriptional gene silencing in which the introduction of a few hundred nucleotide pairs of double-stranded RNA triggers degradation of RNA transcripts containing homologous sequences.

Translation control

Protein modification

12.9 Programmed DNA Rearrangements 1. Gene amplification 2. Antibody and T-cell receptor variability 3. Mating type interconversion 4. Transcriptional control of mating type

Gene amplification Gene amplification: A process in which certain genes undergo differential replication either within the chromosome or extrachromosomally, increasing the number of copies of the gene.

Mutants of ADA

Structure of the immunoglobulin G (IgG) molecule

IgG antibody molecule

Formation of a gene for the light chain of an antibody molecule

Expression of human kappa chain

The variable region of the H chain gene

The level of antibody variability

Life cycle of the yeast

Mating type switching in the yeast

The process of mating type switching in yeast

Genetic basis of mating type interconversion

Transcriptional regulation of mating type in yeast

Structure of the a1/a2 protein bound with DNA

Induction of transcriptional activity by environmental factors (heat shock)

Induction of transcriptional activity by environmental factors (light)

Induction of transcriptional activity by biological factors (steroid hormone)

Induction of transcriptional activity by biological factors (peptide hormones)