How are genes turned on & off? E. coli, a type of bacteria that lives in the human colon Can tune its metabolism to the changing environment and food sources

This metabolic control occurs on two levels Adjusting the activity of metabolic enzymes already present Regulating the genes encoding the metabolic enzymes Figure 18.20a, b (a) Regulation of enzyme activity Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 4 Enzyme 5 Regulation of gene expression Feedback inhibition Tryptophan Precursor (b) Regulation of enzyme production Gene 2 Gene 1 Gene 3 Gene 4 Gene 5 –

Operons: The Basic Concept In bacteria, genes are often clustered into operons, composed of An operator, an “on-off” switch A promoter Genes for metabolic enzymes

An operon Is usually turned “on” Can be switched off by a protein called a repressor

The trp operon: regulated synthesis of repressible enzymes Figure 18.21a (a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the DNA at the promoter and transcribes the operon’s genes. Genes of operon Inactive repressor Protein Operator Polypeptides that make up enzymes for tryptophan synthesis Promoter Regulatory gene RNA polymerase Start codon Stop codon trp operon 5 3 mRNA 5 trpD trpE trpC trpB trpA trpR DNA mRNA E D C B A

Tryptophan present, repressor active, operon off. As tryptophan DNA mRNA Protein Tryptophan (corepressor) Active repressor No RNA made Tryptophan present, repressor active, operon off. As tryptophan accumulates, it inhibits its own production by activating the repressor protein. (b) Figure 18.21b

Repressible and Inducible Operons: Two Types of Negative Gene Regulation In a repressible operon Binding of a specific repressor protein to the operator shuts off transcription In an inducible operon Binding of an inducer to an innately inactive repressor inactivates the repressor and turns on transcription

The lac operon: regulated synthesis of inducible enzymes Figure 18.22a DNA mRNA Protein Active repressor RNA polymerase No RNA made lacZ lacl Regulatory gene Operator Promoter 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) 5 3

lacl mRNA 5' DNA mRNA Protein Allolactose (inducer) Inactive repressor lacz lacY lacA RNA polymerase Permease Transacetylase -Galactosidase 5 3 (b) Lactose present, repressor inactive, operon on. Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced. mRNA 5 lac operon Figure 18.22b

Inducible enzymes Repressible enzymes Usually function in catabolic pathways Repressible enzymes Usually function in anabolic pathways

Regulation of both the trp and lac operons Involves the negative control of genes, because the operons are switched off by the active form of the repressor protein

Positive Gene Regulation Some operons are also subject to positive control Via a stimulatory activator protein, such as catabolite activator protein (CAP)

In E. coli, when glucose, a preferred food source, is scarce The lac operon is activated by the binding of a regulatory protein, catabolite activator protein (CAP) Promoter Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized. If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for the lactose pathway. (a) CAP-binding site Operator RNA polymerase can bind and transcribe Inactive CAP Active CAP cAMP DNA Inactive lac repressor lacl lacZ Figure 18.23a

When glucose levels in an E. coli cell increase CAP detaches from the lac operon, turning it off Figure 18.23b (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized. When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription. Inactive lac repressor Inactive CAP DNA RNA polymerase can’t bind Operator lacl lacZ CAP-binding site Promoter

Eukaryotic Gene Regulation

In eukaryotes, the DNA-protein complex, called chromatin is ordered into higher structural levels than the DNA-protein complex in prokaryotes Figure 19.1

Both prokaryotes and eukaryotes Must alter their patterns of gene expression in response to changes in environmental conditions Eukaryotic DNA Is precisely combined with a large amount of protein Eukaryotic chromosomes Contain an enormous amount of DNA relative to their condensed length

Nucleosomes, or “Beads on a String” Proteins called histones Are responsible for the first level of DNA packing in chromatin Bind tightly to DNA The association of DNA and histones Seems to remain intact throughout the cell cycle

(a) Nucleosomes (10-nm fiber) In electron micrographs Unfolded chromatin has the appearance of beads on a string Each “bead” is a nucleosome The basic unit of DNA packing 2 nm 10 nm DNA double helix Histone tails His- tones Linker DNA (“string”) Nucleosome (“bad”) Histone H1 (a) Nucleosomes (10-nm fiber) Figure 19.2 a

Gene expression can be regulated at any stage, but the key step is transcription All organisms must regulate which genes are expressed at any given time During development of a multicellular organism its cells undergo a process of specialization in form and function called cell differentiation

Differential Gene Expression Each cell of a multicellular eukaryote expresses only a fraction of its genes In each type of differentiated cell, a unique subset of genes is expressed Many key stages of gene expression can be regulated in eukaryotic cells Genes within highly packed chromatin are usually not expressed Figure 19.3 Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethlation Gene DNA Gene available for transcription RNA Exon Transcription Primary transcript RNA processing Transport to cytoplasm Intron Cap mRNA in nucleus Tail CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypetide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein

Regulation of Transcription Initiation 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 (how tightly packed is the chromatin?)

Organization of a Typical Eukaryotic Gene Associated with most eukaryotic genes are multiple control elements Segments of noncoding DNA that help regulate transcription by binding certain proteins Enhancer (distal control elements) Proximal control elements DNA Upstream Promoter Exon Intron Poly-A signal sequence Termination region Transcription Downstream Poly-A signal Primary RNA transcript (pre-mRNA) 5 Intron RNA RNA processing: Cap and tail added; introns excised and exons spliced together Coding segment P G mRNA 5 Cap 5 UTR (untranslated region) Start codon Stop 3 UTR tail Chromatin changes RNA processing degradation Translation Protein processing and degradation Cleared 3 end of primary transport Figure 19.5

The Roles of Transcription Factors To initiate transcription Eukaryotic RNA polymerase requires the assistance of proteins called transcription factors *Enhancers and Specific Transcription Factors: 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

An activator Is a protein that binds to an enhancer and stimulates transcription of a gene Distal control element Activators Enhancer Promoter Gene TATA box General transcription factors DNA-bending protein Group of Mediator proteins RNA Polymerase II RNA synthesis Transcription Initiation complex Chromatin changes RNA processing mRNA degradation Translation Protein processing and degradation A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. 2 Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. 1 The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. 3 Figure 19.6

Some specific transcription factors function as repressors to inhibit expression of a particular gene Some activators and repressors act indirectly by influencing chromatin structure

Coordinately Controlled Genes Unlike the genes of a prokaryotic operon Coordinately controlled eukaryotic genes each have a promoter and control elements The same regulatory sequences Are common to all the genes of a group, enabling recognition by the same specific transcription factors

Mechanisms of Post-Transcriptional Regulation An increasing number of examples are being found of regulatory mechanisms that operate at various stages after transcription In alternative RNA splicing different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Exons DNA Primary RNA transcript RNA splicing or

mRNA Degradation The life span of mRNA molecules in the cytoplasm is an important factor in determining the protein synthesis in a cell &is determined in part by sequences in the leader and trailer regions What happens if genes involved in regulating the cell cycle are turned on/off at the wrong times?