Lecture notes chapter 16 PDF

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Chapter 16: Regulation of Gene Expression How Is Gene Expression Regulated in Prokaryotes? ● Cells make some proteins only when they are needed. To shut off supply of a protein, the cell can: ○ Downregulate mRNA transcription ○ Hydrolyze mRNA, preventing translation ○ Prevent mRNA translation at the ribosome ○ Hydrolyze the protein after it is made ○ Inhibit the proteinʼs function ● The earlier the cell can stop protein synthesis, the less energy is wasted. ● Blocking transcription is more efficient than transcribing the gene, translating the message, and then degrading or inhibiting the protein. ● Gene expression begins at the promoter. ● Two types of regulatory proteins can bind to promoters: ○ Negative regulators—a repressor protein prevents transcription ○ Positive regulators—an activator protein stimulates transcription ● Positive and Negative Regulation ○ Gene is normally transcribed, but binding of a repressor protein prevents transcription.

● E. coli in the human intestine must adjust quickly to changes in food supply. ● Glucose is the preferred energy source, but in absence of glucose/presence of lactose, E. coli will switch to lactose metabolism. ● Three proteins are needed for the uptake and metabolism of lactose: ○ β -galactoside permease—carrier protein that moves lactose into the cell ○ β -galactosidase—hydrolyses lactose (a-β -galactoside--disaccharide of galactose and glucose) ○ β -galactoside transacetylase—transfers acetyl groups from acetyl CoA to certain b-galactosides ● If E.coli is grown with glucose but no lactose, no enzymes for lactose conversion

are produced. ● If lactose is predominant and glucose is low, E.coli synthesizes b-galactosidase (and the other two enzymes) after a short lag period. Why the lag? An Inducer Stimulates the Expression of a Gene for an Enzyme

Two Ways to Regulate a Metabolic Pathway

The lac Operon of E. coli ● The three genes for lactose metabolism share a promoter, and are transcribed together. ● A gene cluster with a single promoter is an operon—the one that encodes the lactose enzymes is the lac operon. ● A typical operon consists of: ○ A promoter ○ Two or more structural genes ○ An operator—a short stretch of DNA between the promoter and the structural genes.

● Several ways to control operon transcription. Examples: ○ An inducible operon regulated by a repressor protein ○ A repressible operon regulated by a repressor protein ○ An operon regulated by an activator protein The lac Operon: An Inducible System ● The lac operon has: ○ A promoter that binds RNA polymerase to initiate transcription ○ An operator that binds a repressor protein, which blocks transcription ● An activator protein can increase transcription through positive control

● ● If high glucose, then low cAMP, and CRP does not bind to the lac operon promoter--efficiency of transcription is reduced. ● Presence of a preferred energy source (glucose) represses other catabolic pathways. Potential Points for the Regulation of Eukaryotic Gene Expression ● Eukaryotic gene expression must be regulated to ensure proper timing and location of protein production. ● Regulation can and does occur at multiple points before/during/following transcription and translation.

The Initiation of Transcription in Eukaryotes ● Several transcription factors (regulatory proteins) must assemble on the chromosome before RNA polymerase can bind to the promoter. ● TFIID binds to the TATA box; then other transcription factors and RNA polymerase II bind, forming a transcription complex.

Transcription Factors, Repressors, and Activators

How Is Eukaryotic Gene Transcription Regulated? ● Transcription factors have common structural motifs in the domains that bind to DNA.

Protein–DNA Interactions ● Three general criteria for DNA recognition by a protein motif: ○ Fits into major or minor groove ○ Has amino acids that can project into interior of double helix ○ Has amino acids that can bond with interior bases

Expression of Specific Transcription Factors ● Turns Fibroblasts into Neurons During development, cell differentiation is often mediated by changes in gene expression. ● All differentiated cells contain the entire genome; their specific characteristics arise from differential gene expression. ● Cellular therapy is a new approach to diseases that involve degeneration of one cell type. ● Alzheimerʼs disease involves degeneration of neurons in the brain. ● If other cells from the same individual could be made to differentiate into neurons (as shown here, with fibroblasts), they could theoretically be

transferred to the patient. ● More on “resetting” cells later. Coordinating Gene Expression ● Eukaryotic genes that are regulated simultaneously may be far apart or on different chromosomes. ● Gene expression is coordinated if they have the same regulatory sequences that bind same transcription factors. ● For example: A regulatory sequence in plant genes called the stress response element (SRE)—found near genes coding for proteins needed to cope with, among other conditions, drought.

How Do Viruses Regulate Their Gene Expression? ● Virus particles, called virions, are acellular, usually composed of only nucleic acid and a few proteins. ● Viruses use host cells to carry out metabolic functions while they reproduce. ● Studying their reproductive cycle has uncovered principles of gene expression and regulation. Bacteriophage and Host

● The lytic cycle is a typical viral reproductive cycle—the host cell lyses and releases virus particles. ● A phage injects a host cell with nucleic acid that takes over synthesis. ● New phage particles appear rapidly and are soon released from the lysed cell.

The Lytic Cycle: A Strategy for Viral

The Lytic and Lysogenic Cycles of Bacteriophage

Control of Bacteriophage l Lysis and Lysogeny ● A genetic switch senses the host’s condition; two regulatory proteins—c1 (“c one”) and Cro—compete for promoters on the phage DNA. ● The two promoters control viral gene transcription and the regulatory proteins have opposite effects on each promoter. ● c1 and Cro are made early in phage infection and there is essentially a competition between them. ● In a rapidly growing host, Cro synthesis is low and c1 protein accumulates—the phage enters a lysogenic cycle. ● Under stressful conditions, c1 is degraded, more Cro is produced, and genes for lysis are activated.

How Do Epigenetic Changes Regulate Gene Expression? ● Epigenetics refers to changes in expression in a gene or set of genes, without a change in the DNA sequence. ● Changes are reversible, but sometimes heritable and stable. ● Includes two processes: DNA methylation and chromosomal protein alterations. DNA Methylation: an Epigenetic Change ● Some cytosine residues in DNA are modified by covalently adding a methyl group. ● DNA methyltransferase catalyzes the reaction—usually in adjacent C and G residues. ● Regions are called CpG islands— often in promoters. ● When DNA replicates, a maintenance methylase catalyzes formation of 5′methylcytosine in the new strand. ● Methylation pattern may be altered. ● Demethylase can catalyze the removal of the methyl group.

How Do Epigenetic Changes Regulate Gene Expression? ● Effects of DNA methylation: ○ Methylated DNA attracts proteins that are involved in repression of transcription and can inactivate DNA ○ Important in development—early demethylation allows many genes to become active ○ In cancer, misregulation can occur in oncogenes and tumor suppressors Epigenetic Remodeling of Chromatin for Transcription ● Chromatin remodeling is the alteration of chromatin structure. ● Nucleosomes contain DNA and histones in a tight complex, inaccessible to RNA polymerase. ● Each histone has a positively charged “tail” at its N terminus. ● Histone acetyltransferases change the tail’s charge by adding acetyl groups to the amino acids (less “+” so lower affinity for “-” charge on DNA.

● Acetylation opens up nucleosomes and activates transcription.

How Do Epigenetic Changes Regulate Gene Expression? ● Environmentally-caused epigenetic changes can be inherited. ● If a germline cell that forms gametes is changed, changes can be passed on. ● Twin studies show that the environment can produce different epigenetic modifications, and thus differences in gene regulation in genetically identical individuals. ● Environmental factors that may lead to epigenetic changes: ○ Stress—genes become heavily methylated ○ Addiction or psychosis may produce different DNA methylation or histone modification patterns Genomic Imprinting

X Chromosome Inactivation ● Patterns of DNA methylation may include large regions or whole chromosomes. ● Two kinds of chromatin are visible during interphase: ○ Euchromatin—diffuse and light-staining; contains DNA for mRNA transcription ○ Heterochromatin—condensed, dark-staining, contains genes not transcribed ● A type of heterochromatin is the inactive X chromosome in mammals. ● Males (XY) and females (XX) contain different numbers of X-linked genes, yet for most genes transcription rates are similar. ● Early in development, one of the X chromosomes is inactivated. ● The inactivated X chromosome remains in the nucleus. ● This Barr body is identifiable during interphase and can be seen in cells of human females. ● The Barr body is a lump of heterochromatin, and consists of heavily methylated DNA, except for one gene, Xist. ● On the inactive X chromosome Xist is lightly methylated and active. ● RNA transcribed from Xist binds to the chromosome, spreading inactivation— an example of interference RNA.

Euchromatin and Heterochromatin

How Is Eukaryotic Gene Expression Regulated After Transcription? ● Eukaryotic gene expression can be regulated in the nucleus before mRNA export, or after mRNA leaves. ● Control mechanisms include: ○ alternative splicing of pre-mRNA ○ microRNAs ○ translation repressors ○ regulation of protein breakdown Alternative Splicing Results in Different ● Mature mRNAs and Proteins Different mRNAs can be made from the same gene by alternative splicing. ● As introns and exons are spliced out, new proteins are made. ● A mechanism for generating proteins with different functions, from a single gene.

mRNA Inhibition by mi- and siRNAs ● MicroRNAs(miRNAs)—small molecules of noncoding RNA—are important regulators of gene expression. ● Each miRNA is about 22 bases long and has many targets. ● miRNAs are transcribed as longer precursors then cleaved to miRNAs. ● Proteins guide miRNA to target mRNA—translation is inhibited and mRNA is degraded. ● Small interfering RNAs (siRNAs) also result in RNA silencing. ● Often arise from viral infection and transposon sequences. They bind to target mRNA and cause its degradation. ● May have evolved as defense to prevent translation of viral and transposon sequences. mRNA translation can be regulated ● Protein and mRNA concentrations are not consistently related— governed by factors acting after mRNA is made. ● Cells can block mRNA translation or alter how long new proteins persist in the cell.

● Multiple ways to regulate mRNA translation, including: ○ miRNAs can inhibit translation ○ Repressor proteins can block translation directly

A Proteasome Breaks Down Proteins ● Protein longevity is regulated— protein content is a function of synthesis and degradation. ● Ubiquitin attaches to a protein marked for destruction and attracts other ubiquitins. ● This complex binds to a proteasome—a large complex where the ubiquitin is removed and the protein is digested....