Regulation of Gene Expression From: University of Wisconsin, Department of Biochemistry

Potential Points for the Regulation of Gene Expression

Bacteria often respond to environmental change by regulating transcription Natural selection has favored bacteria that produce only the products needed by that cell A cell can regulate the production of enzymes by gene regulation Gene expression in bacteria is controlled by the operon model Prokaryotes

Repressible and Inducible Operons: Two Types of Negative Gene Regulation 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 An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription (lac operon ) Transcriptional Control

Fig. 18-3a Polypeptide subunits that make up enzymes for tryptophan synthesis Tryptophan absent, repressor inactive, operon on DNA mRNA 5 ProteinInactive repressor RNA polymerase Regulatory gene Promoter trp operon Genes of operon Operator Stop codon Start codon mRNA trpA 5 3 trpRtrpE trpD trpCtrpB AB CD E The trp operon is a repressible operon

Fig. 18-3b-1 Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA

Fig. 18-3b-2 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA

By itself, the lac repressor is active and switches the lac operon off A molecule called an inducer inactivates the repressor to turn the lac operon on The lac operon is inducible.

Fig. 18-4a DNA Protein Active repressor RNA polymerase Regulatory gene Promoter Operator mRNA 5 3 No RNA made lac I lacZ Lactose absent, repressor active, operon off

Fig. 18-4b Lactose present, repressor inactive, operon on mRNA Protein DNA mRNA 5 Inactive repressor Allolactose (inducer) 5 3 RNA polymerase Permease Transacetylase lac operon  -Galactosidase lacY lacZlacAlac I

Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP

Fig cAMP DNA Inactive lac repressor Allolactose Inactive CAP lac I CAP-binding site Promoter Active CAP Operator lacZ RNA polymerase binds and transcribes Inactive lac repressor lacZ Operator Promoter DNA CAP-binding site lac I RNA polymerase less likely to bind Inactive CAP Lactose present, glucose present (cAMP level low): little lac mRNA synthesized Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized CAP interaction with cAMP animation

Organization of a Typical Eukaryotic Gene Associated with most eukaryotic genes are control elements, segments of noncoding DNA that help regulate transcription by binding certain proteins Control elements and the proteins they bind are critical to the precise regulation of gene expression in different cell types James Darnell chats briefly about signal molecules

Fig Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Intron Organization of a Typical Eukaryotic Gene Control elements are segments of noncoding DNA that help regulate transcription by binding certain proteins HHMI transcription factor interaction animation

Fig Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Intron Cleaved 3 end of primary transcript Primary RNA transcript Poly-A signal Transcription 5 Exon Intron

Fig Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Intron Exon Intron Cleaved 3 end of primary transcript Primary RNA transcript Poly-A signal Transcription 5 RNA processing Intron RNA Coding segment mRNA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail 3

A Transcription Factor Protein Binds to DNA Transcription factors recognize particular nucleotide sequences: NFATs (nuclear factors of activated T cells) are transcription factors that control genes in the immune system. They bind to a recognition sequence near the genes’ promoters. The binding produces an induced fit—the protein changes conformation. How single molecules move animation

Fig Enhancer TATA box Promoter Activators DNA Gene Distal control element An activator is a protein that binds to an enhancer and stimulates transcription of a gene

Fig Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors Bound activators cause mediator proteins to interact with proteins at the promoter Transcription factors act at eukaryotic promoters.

Fig Enhancer TATA box Promoter Activators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

Fig Control elements Enhancer Available activators Albumin gene (b) Lens cell Crystallin gene expressed Available activators LENS CELL NUCLEUS LIVER CELL NUCLEUS Crystallin gene Promoter (a) Liver cell Crystallin gene not expressed Albumin gene expressed Albumin gene not expressed

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 Some other processes promoting differential gene expression: – Histone modification – DNA methylation Eukaryotes

A chromosome consists of a DNA molecule packed together with proteins © 2011 Pearson Education, Inc. DNA double helix (2 nm in diameter) DNA, the double helix Nucleosome (10 nm in diameter) Histones Histone tail H1 Nucleosomes, or “beads on a string” (10-nm fiber)

Figure 16.22b 30-nm fiber Loops Scaffold 300-nm fiber Chromatid (700 nm) Replicated chromosome (1,400 nm) Looped domains (300-nm fiber) Metaphase chromosome

Histone tails DNA double helix (a) Histone tails protrude outward from a nucleosome Acetylated histones Amino acids available for chemical modification (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Histone Modifications In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails

DNA methylation, the addition of methyl groups to certain bases in DNA, is associated with reduced transcription in some species DNA Methylation: An Epigenetic Change

Methylated DNA binds proteins that are involved in repression of transcription— genes tend to be inactive (silenced). Patterns of DNA methylation may include large regions or whole chromosomes. Effects of DNA Methylation:

A Barr body is the transcriptionally inactive X chromosome A Barr body consists of heavily methylated DNA

Summary: Epigenetic Inheritance Chromatin modifications do not alter DNA sequence, but 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 Monozygotic twins show different DNA methylation patterns after living in different environments. From: The Proceedings of the National Academy of Sciences: The image shows methylation patterns for three-year-old twins (left) and 50-year-old twins (right), with the differences highlighted in red.

or RNA splicing mRNA Primary RNA transcript Troponin T gene Exons DNA Alternative RNA Splicing

mRNA Degradation Nucleotide sequences that influence the lifespan of mRNA in eukaryotes reside in the untranslated region (UTR) at the 3 end of the molecule

Noncoding RNAs play multiple roles in controlling gene expression Only a small fraction of DNA codes for proteins, rRNA, and tRNA A significant amount of the genome may be transcribed into noncoding RNAs Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration

Micro RNAs (miRNAs) Figure Degradation of mRNA OR Blockage of translation Target mRNA miRNA Protein complex Dicer Hydrogen bond The micro- RNA (miRNA) precursor folds back on itself, held together by hydrogen bonds. 1 2 An enzyme called Dicer moves along the double- stranded RNA, cutting it into shorter segments. 2 One strand of each short double- stranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins. 3 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence. 4 The miRNA-protein complex prevents gene expression either by degrading the target mRNA or by blocking its translation. 5 Small single-stranded RNA molecules that can bind to mRNA degrading it or blocking its translation MicroRNA animation (NATURE)

Proteasome and ubiquitin to be recycled Proteasome Protein fragments (peptides) Protein entering a proteasome Ubiquitinated protein Protein to be degraded Ubiquitin Protein Processing and Degradation Proteasomes are giant protein complexes that bind protein molecules and degrade them The length of time a protein functions before it is degraded is strictly regulated. HHMI Animation

Fig. 18-UN4 Genes in highly compacted chromatin are generally not transcribed. Chromatin modification DNA methylation generally reduces transcription. Histone acetylation seems to loosen chromatin structure, enhancing transcription. Chromatin modification Transcription RNA processing Translation mRNA degradation Protein processing and degradation mRNA degradation Each mRNA has a characteristic life span, determined in part by sequences in the 5 and 3 UTRs. Protein processing and degradation by proteasomes are subject to regulation. Protein processing and degradation Initiation of translation can be controlled via regulation of initiation factors. Translation ormRNA Primary RNA transcript Alternative RNA splicing: RNA processing Coordinate regulation: Enhancer for liver-specific genes Enhancer for lens-specific genes Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription. Transcription Regulation of transcription initiation: DNA control elements bind specific transcription factors.

What about cell differentiation and differential gene expression? During embryonic development, a fertilized egg gives rise to many different cell types (a) Cytoplasmic determinants in the egg Unfertilized egg Sperm Fertilization Zygote (fertilized egg) Mitotic cell division Two-celled embryo Nucleus Molecules of two different cytoplasmic determinants

How do tissues develop? Interactions between cells induce differentiation of specialized cell types Induction: signal molecules from embryonic cells cause transcriptional changes in nearby target cells Induction by nearby cells Early embryo (32 cells) NUCLEUS Signal transduction pathway Signal receptor Signaling molecule (inducer)

GHOSTS

Sequential Regulation of Gene Expression During Cellular Differentiation Determination commits a cell to its final fate Determination precedes differentiation Cell differentiation is marked by the production of tissue-specific proteins

Fig Embryonic precursor cell Nucleus OFF DNA Master regulatory gene myoD Other muscle-specific genes OFF

Fig Embryonic precursor cell Nucleus OFF DNA Master regulatory gene myoD Other muscle-specific genes OFF mRNA MyoD protein (transcription factor) Myoblast (determined)

Fig Embryonic precursor cell Nucleus OFF DNA Master regulatory gene myoD Other muscle-specific genes OFF mRNA MyoD protein (transcription factor) Myoblast (determined) mRNA Myosin, other muscle proteins, and cell cycle– blocking proteins Part of a muscle fiber (fully differentiated cell) MyoD Another transcription factor

DNA Methylation: An Epigenetic Change

Many Prokaryotic Genes Are Regulated in Operons Sigma factors—other proteins that bind to RNA polymerase and direct it to specific promoters