Regulation of Gene Expression Chapter 18 Regulation of Gene Expression

Overview: Conducting the Genetic Orchestra Prokaryotes and eukaryotes alter gene expression in response to their changing environment In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types

Eye Leg Antenna Wild type Mutant Figure 18.20 Abnormal pattern formation in Drosophila. Antenna Wild type Mutant

Bacteria regulate their gene expression Feedback mechanisms allow control over metabolism so that cells produce only the products needed at that time (with some limitations) This metabolic control occurs on two levels: 1. adjusting activity of metabolic enzymes 2. regulating genes that encode metabolic enzymes

Regulation of a Metabolic Pathway Precursor Feedback inhibition trpE gene Enzyme 1 In the pathway for tryptophan synthesis, an abundance of tryptophan can both inhibit the activity of the first enzyme in the pathway (feedback inhibition), a rapid response, and repress expression of the genes for all the enzymes needed for the pathway, a longer-term response. trpD gene Regulation of gene expression Enzyme 2 trpC gene trpB gene Enzyme 3 Figure 18.2 Regulation of a metabolic pathway trpA gene Tryptophan Regulation of enzyme activity (b) Regulation of enzyme production

Polypeptides (5) that make up enzymes for tryptophan synthesis In bacteria, genes are often clustered into operons, composed of: 1. an operator, an “on-off” switch 2. a promoter 3. genes for metabolic enzymes An operon can be switched off by a protein called a repressor binds only to the operator trp operon Promoter Promoter Genes of operon (5) DNA trpR trpE trpD trpC trpB trpA Operator Regulatory gene no tryptophan RNA polymerase Stop codon Start codon 3¢ mRNA 5¢ repressor inactive mRNA 5¢ E D C B A operon ON Polypeptides (5) that make up enzymes for tryptophan synthesis Protein Repressor (inactive) Tryptophan absent, repressor inactive, operon on

Operons A repressible operon- is one that is usually on; binding of a repressor to the operator shuts off transcription the trp operon is a repressible operon repressible enzymes usually function in anabolic pathways cells suspend production of an end product that is not needed

The trp operon operon OFF corepressor- active tryptophan DNA DNA No RNA made mRNA mRNA Protein Protein Active repressor Active repressor corepressor- a small molecule that cooperates with a repressor to switch an operon off Tryptophan (corepressor) Tryptophan (corepressor) Tryptophan present, repressor active, operon off

Operons A repressible operon- is one that is usually on; binding of a repressor to the operator shuts off transcription the trp operon is a repressible operon repressible enzymes usually function in anabolic pathways cells suspend production of an end product that is not needed An inducible operon- is one that is usually off; a molecule, an inducer, inactivates the repressor & turns on transcription the classic example of an inducible operon is the lac operon inducible enzymes usually function in catabolic pathways cells only produce enzymes when there’s a nutrient that needs to be broken down

The lac operon operon OFF Regulatory gene no lactose repressor active Promoter Operator DNA lacl lacZ No RNA made no lactose 3¢ mRNA RNA polymerase repressor active 5¢ operon OFF Active repressor Protein Lactose absent, repressor active, operon off The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator.

a small molecule that inactivates the repressor operon ON Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced. lac operon 3 genes DNA lacl lacZ lacY lacA RNA polymerase 3¢ mRNA mRNA 5¢ 5¢ Permease Transacetylase Protein -Galactosidase E.Coli uses 3 enzymes to take up and metabolize lactose Inactive repressor Allolactose (inducer) β-galactosidase: hydrolyzes lactose to glucose and galactose permease: transports lactose into the cell transacetylase: function in lactose metabolism is still unclear Lactose present, repressor inactive, operon on inducer- a small molecule that inactivates the repressor operon ON repressor inactive lactose

Gene Regulation Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor Some operons are also subject to positive control through a stimulatory activator protein, such as catabolite activator protein (CAP) The lac operon is under dual control: negative control by the lac repressor positive control by CAP

Positive Control lacl lacZ glucose cAMP When glucose (a preferred food source of E. coli ) is scarce, the lac operon is activated by the binding of CAP Promoter DNA lacl lacZ RNA polymerase can bind and transcribe Operator CAP-binding site glucose cAMP Active CAP cAMP Inactive lac repressor Inactive CAP Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized

When glucose levels increase, CAP detaches from the lac operon, turning it off Promoter DNA lacl lacZ CAP-binding site Operator RNA polymerase can’t bind Inactive CAP Inactive lac repressor Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

REVIEW Repressible Operon Inducible Operon Genes expressed Genes not expressed Promoter Genes Operator Active repressor: corepressor bound Inactive repressor: no corepressor present Corepressor Inducible Operon Genes not expressed Genes expressed Fig. 18-UN3 Promoter Operator Genes Active repressor: no inducer present Inactive repressor: inducer bound

Eukaryotic Genomes Two features of eukaryotic genomes are a major information-processing challenge: 1. the typical eukaryotic genome is much larger than that of a prokaryotic cell 2. cell specialization limits the expression of many genes to specific cells The DNA-protein complex, called chromatin, is ordered into higher structural levels than the DNA-protein complex in prokaryotes

Differential Gene Expression Almost all the cells in an organism are genetically identical Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome Errors in gene expression can lead to diseases including cancer

***** most important control point Gene Expression Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethylation #1 Gene expression is regulated at many stages DNA Gene available for transcription Gene #2 ***** most important control point Transcription RNA Exon Primary transcript Intron #3 RNA processing Tail Cap mRNA in nucleus Transport to cytoplasm CYTOPLASM #4 mRNA in cytoplasm #5 Degradation of mRNA Translation Figure 18.6 Stages in gene expression that can be regulated in eukaryotic cells. Polypeptide Protein processing, such as cleavage and chemical modification #6 #6 Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity, structural support)

#1 Histones Genes within highly packed heterochromatin (highly condensed areas) are usually not expressed Chemical modifications to histones and DNA of chromatin influence both chromatin structure and gene expression histone acetylation- acetyl groups (-COCH3) are attached to positively charged lysines in histone tails This process seems to loosen chromatin structure, thereby promoting the initiation of transcription #1 #2 #3 #4 #5 #6 Histone tails DNA double helix Amino acids available for chemical modification Histone tails protrude outward from a nucleosome Unacetylated histones Acetylated histones Acetylation of histone tails promotes loose chromatin structure that permits transcription

DNA Methylation DNA methylation- #1 DNA Methylation DNA methylation- the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species DNA methylation can cause long-term inactivation of genes in cellular differentiation In genomic imprinting, methylation turns off either the maternal or paternal alleles of certain genes at the start of development

Summary- Chromatin Modifications #1 Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery

(distal control elements) Eukaryotic Gene #2 & #3 Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins Control elements & the proteins they bind are critical to the precise regulation of gene expression in different cell types Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Exon Intron Exon Intron Exon DNA Upstream Downstream Promoter Transcription Poly-A signal Primary RNA transcript (pre-mRNA) Exon Intron Exon Intron Exon Cleaved 3¢ end of primary transcript #1 5¢ #2 RNA processing: Cap and tail added; introns excised and exons spliced together #3 Intron RNA #4 #5 Coding segment #6 mRNA 3¢ Start codon Stop codon 5¢ Cap 5¢ UTR (untranslated region) 3¢ UTR (untranslated region) Poly-A tail

To initiate transcription, eukaryotic RNA polymerase requires the #2 To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors (TFs) General TFs are essential for the transcription of all protein-coding genes In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific TFs proximal control elements- are located close to the promoter distal control elements- groups of which are called enhancers, may be far away from a gene or even in an intron activator- specific TF, a protein that binds to an enhancer & stimulates transcription of a gene Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. repressor- specific TF, inhibit expression of a gene

Combinatorial Control of Gene Activation #2 Enhancer Promoter Control elements Albumin gene A particular combination of control elements can activate transcription only when the appropriate activator proteins are present Crystallin gene LIVER CELL NUCLEUS LENS CELL NUCLEUS Available activators Available activators Albumin gene not expressed Figure 18.11 Cell type–specific transcription. Albumin gene expressed Crystallin gene not expressed Crystallin gene expressed (a) Liver cell (b) Lens cell

Coordinately Controlled Genes #2 Coordinately Controlled Genes Unlike the genes of a prokaryotic operon, each of the co-expressed eukaryotic genes has a promoter and control elements These genes can be scattered over different chromosomes, but each has the same combination of control elements Copies of the activators recognize specific control elements and promote simultaneous transcription of the genes

Mechanisms of Post-Transcriptional Regulation #3-6 Mechanisms of Post-Transcriptional Regulation Transcription alone does not account for gene expression More and more examples are being found of regulatory mechanisms that operate at various stages after transcription Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes

Alternative RNA Splicing #3 Exons DNA Troponin T gene Primary RNA transcript Figure 18.11 Alternative RNA splicing of the troponin T gene different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons/introns RNA splicing mRNA or

mRNA Degradation #4 The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis Eukaryotic mRNA is more long lived than prokaryotic mRNA Nucleotide sequences that influence the lifespan of mRNA in eukaryotes reside in the untranslated region (UTR) at the 3’ end of the molecule

Initiation of Translation #5 The initiation of translation of selected mRNAs can be blocked by regulatory proteins that bind to specific sequences or structures of the mRNA Alternatively, translation of all mRNAs in a cell may be regulated simultaneously For example, translation initiation factors are simultaneously activated in an egg following fertilization

Protein Processing and Degradation #6 After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control #1 proteasomes- are giant protein complexes that bind protein molecules and degrade them #2 #3 #4 #5 Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. Multiple ubiquitin molecules are attached to a protein by enzymes in the cytosol. The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity. #6 Ubiquitin Proteasome and ubiquitin to be recycled Proteasome Protein to be degraded Ubiquitinated protein Protein fragments (peptides) Protein entering a proteasome

REVIEW Figure 18.UN04 Summary figure, Concept 18.2 Chromatin modification Transcription • Genes in highly compacted chromatin are generally not transcribed. • Regulation of transcription initiation: DNA control elements in enhancers bind specific transcription factors. • Histone acetylation seems to loosen chromatin structure, enhancing transcription. Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. • DNA methylation generally reduces transcription. • Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Chromatin modification Transcription RNA processing • Alternative RNA splicing: RNA processing Primary RNA transcript mRNA degradation Translation mRNA or Figure 18.UN04 Summary figure, Concept 18.2 Protein processing and degradation Translation • Initiation of translation can be controlled via regulation of initiation factors. mRNA degradation • Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Protein processing and degradation • Protein processing and degradation by proteasomes are subject to regulation.

Noncoding RNAs play multiple roles in controlling gene expression Only a small fraction of DNA codes for proteins, and a very small fraction of the non-protein-coding DNA consists of genes for RNA such as rRNA and tRNA A significant amount of the genome may be transcribed into noncoding RNAs (ncRNAs) Noncoding RNAs regulate gene expression at two points: mRNA translation chromatin configuration

small single-stranded RNA molecules that can bind to mRNA Hairpin Hydrogen bond miRNA Dicer 5 3 (a) Primary miRNA transcript miRNA miRNA- protein complex MicroRNAs (miRNAs)- small single-stranded RNA molecules that can bind to mRNA These can degrade mRNA or block its translation Figure 18.15 Regulation of gene expression by miRNAs. mRNA degraded Translation blocked (b) Generation and function of miRNAs

RNA interference (RNAi) RNAi is caused by The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi) RNAi is caused by small interfering RNAs (siRNAs) siRNAs and miRNAs are similar but form from different RNA precursors In some yeasts siRNAs play a role in heterochromatin formation and can block large regions of the chromosome RNA-based mechanisms may also block transcription of single genes

Chromatin modification REVIEW Chromatin modification • Small or large noncoding RNAs can promote the formation of heterochromatin in certain regions, blocking transcription. Chromatin modification Transcription Translation RNA processing • miRNA or siRNA can block the translation of specific mRNAs. mRNA degradation Translation Protein processing and degradation Figure 18.UN05 Summary figure, Concept 18.3 mRNA degradation • miRNA or siRNA can target specific mRNAs for destruction.