Regulation of Gene Expression Defects in gene regulation can alter the development of an organism

Seven processes that affect the steady-state concentration of a protein in a cell

Regulation of Gene Expression Principles of gene regulation Regulation of gene expression in prokaryotes Regulation of gene expression in eukaryotes

Principles of Gene Regulation constitutive vs. regulated gene expression housekeeping genes, gene products that are required at all times at a more or less constant level. e.g., in citric acid cycle

Principles of Gene Regulation 1) RNA polymerase binds to DNA at promoters

Principles of Gene Regulation (cont’d) 2) Transcription initiation is regulated by proteins that bind to or near promoters. Repression of a repressible gene:(i.e., negative regulation) repressors (vs. activators) bind to operators of DNA. Repressor is regulated by an effector, usually a small molecule or a protein, that binds and causes a conformational change. Activator binds to DNA sites called enhancer to enhance the RNA polymerase activity. (i.e., positive regulation) Induction of an inducible gene, e.g., heat-shock genes. Heat-shock promoters

Principles of Gene Regulation (cont’d) 3) Most prokaryotic genes are regulated in units called operons. Francois Jacob & Jacques Monod, 1960

Lactose metabolism in E. coli

4) The lac operon is subject to negative regulation: repressor tetrameric repressor IPTG induced uninduced

5) Regulatory proteins have discrete DNA-binding domains Functional groups (pink) in DNA available for protein binding

5) Regulatory proteins have discrete DNA-binding domains e.g., specific amino acid-base pair interactions in DNA-protein interaction

e.g., a DNA-binding domain (3) interacts directly with DNA at major groove

The DNA binding sites for regulatory proteins are often inverted repeats of a short DNA sequence (a palindrome) at which multiple subunits (usually two) of a regulatory protein bind cooperatively. inverted repeats e.g., Lac repressor vs. operator AATTGT…ACAATT TTAACA…TGTTAA

Examples of DNA-binding motifs/domains: helix-turn-helix: e.g., Lac repressor zinc finger: e.g., Zif 268 homeodomain: e.g., Ultrabithorax (Ubx)

Helix-turn-helix Lac repressor, DNA-binding domains a tetramer allolactose-binding domains hydrogen-bonding (red) hydrophobic interactions (yellow)

zinc finger: In many eukaryotic (few prokaryotic) DNA-binding proteins e.g., Zif 268 ~30 a.a. Zn2+ (2 Cys, 2 His)

homeodomain: homeobox: DNA sequence encoding homeodomain e.g., Ultrabithorax (Ubx) a a helix (red) protruding into the major groove

Leucine zippers 6) Regulatory proteins also have protein-protein interaction domains Leucine zippers basic helix-loop-helix interacting Leu (red) Leucine zippers

basic helix-loop-helix e.g., transcription factor Max (dimeric) A pair of interacting Leu helix-loop-helix (red & purple) DNA-binding segment (pink)

Regulation of Gene Expression Principles of gene regulation Regulation of gene expression in prokaryotes Regulation of gene expression in eukaryotes

The Lac Operon

The lac Operon Is Subject to Positive Regulation: Activation by CRP (cAMP receptor protein)

CRP homodimer DNA is bended Region interacting with RNA polymerase (yellow) cAMP (pink)

The effect of glucose on CRP is mediated by cAMP. Transcription occurs only at low glucose and high lactose. cAMP & CRP are involved in the coordinated regulation of many operons. A net of operons with a common regulator is called regulon.

The ara operon undergoes both positive & negative regulation by a single regulatory protein AraC. Th end product of the arabinose metabolic pathway, D-xylulose 5-phosphate, is an intermediate in the pentose phosphate pathway.

When the AraC repressor is depleted, The araC gene is transcribed from its own promoter.

At high glucose and low arabinose, AraC binds and brings araO2 and araI sites together to form a DNA loop, repressing araBAD.

At low glucose, but arabinose is present, AraC repressor binds arabinose and changes conformation to become an activator. DNA loop is opened, and AraC binds to each half-site of araI and araO1. The proteins interact with each other, and act in concert with CRP-cAMP to facilitate transcription of the araBAD genes.

Many Genes for Amino Acid Biosynthesis Are Regulated by Transcription Attenuation e.g., the trp operon At high tryptophen, 1) the repressor binds its operator, 2) transcription of trp mRNA is attenuated.

Trp repressor dimeric, helix-turn-helix bound tryptophen (red)

Transcription attenuation in the trp operon The trp mRNA leader (trpL): Sequence 1 encodes a small peptide, leader peptide, containing two Trp residues.

Transcription attenuation in the trp operon attenuator At high trp At low trp

The Trp Operon

Induction of the SOS Response in E. coli Requires Destruction of Repressor Protein LexA: Operon-like regulation Coprotease RecA is activated by DNA damage (single stranded DNA) LexA is cleaved and inactivated by RecA

Synthesis of Ribosomal Proteins Is Coordinated with rRNA Synthesis mRNAs of some ribosomal proteins (r-protein): r-protein acts as a translational repressor yellow: RNA pol subunits blue: EFs

Synthesis of Ribosomal Proteins Is Coordinated with rRNA Synthesis e.g., stringent response in E. coli, response to amino acid starvation uncharged tRNA binding > stingent factor (RelA) binding > catalysing ppGpp synthesis > binding to b-subunit of pol > rRNA synthesis reduced

Some Genes Are Regulated by Genetic Recombination e.g., regulation of flagellin genes in Salmonella: phase variation allows evasion of host immune response. repressor

Regulation of Gene Expression Principles of gene regulation Regulation of gene expression in prokaryotes Regulation of gene expression in eukaryotes

Extraordinary complexity of gene regulation in eukaryotes Transcriptional Active Chromatin is Structurally Different from Inactive Chromatin: hypersensitive sites (100 ~ 200 bp), DNaseI sensitive sequences whithin the 1000 bp flanking the 5’ end of transcribed genes. Modifications Increase the Accessibility of DNA: e.g., 5’-methylation of cytosine of CpG sequences is common in eukaryotic DNA, active genes tend to be undermethylated. ……

Extraordinary complexity of gene regulation in eukaryotes Chromatin Is Remodeled by Acetylation and Nucleosome Displacements chromatin remodeling: the detailed mechanisms for transcription-associated structure changes in chromatin.

Extraordinary complexity of gene regulation in eukaryotes Many Eukaryotic Promoters Are Positively Regulated DNA-Binding Transactivators and Coactivators Facilitate Assembly of the General Transcription Factors enhancer in higher eukaryotes, upstream activator sequences (UASs) in yeast.

Three Classes of Proteins Are Involved in Transcriptional Activation basal transcription factors, DNA-binding transactivators, and coactivators.

A wide variety of repressors function by a range of mechanisms

The Genes Required for Galactose Metabolism in Yeast Are Subject to Both Positive and Negative Regulation Binding of galactose to Gal3p and its interaction with Gal80p produce a conformation change in Gal80p that allows Gal4p to function in transcription activation. regulated 6 genes (table 28-3) regulatory proteins: Gal4p, Gal80p & Gal3p

Unlike bacteria, there is no operons in yeast. Each of the GAL genes is transcribed separately.

The GAL system is shown to illustrate the transcription activation of a group of related eukaryotic genes. The initiation complexes assemble stepwise: DNA-binding transactivators Basal transcription factors/pol II Additional protein complexes needed to remodel the chromatin e.g., SWI/SNF: histone remodeling SAGA: histone acetylation

Typical DNA-binding transactivators have a DNA-binding domain and an activation domain. e.g., Gal4p, acidic activation domain function in activation CTF1(CCAAT-binding transcription factor 1), proline-rich activation domain Sp1, glutamine-rich activation domain

“Domain-swapping” experiment: A chimeric protein containing the DNA-binding domain of Sp1 and the activation domain of CTF1 activates transcription if a GC box is present.

Eukaryotic Gene Expression Can Be Regulated by Intercellular and Intracellular Signals e.g., steroid hormone (estrogen) receptors

Regulation Can Occur through Phosphorylation of Nuclear Transcription Factors e.g., The catalytic subunit of protein kinase A, released when cAMP levels rise, enters the nucleus and phosphorylates a nuclear protein, the CRE-binding protein (CREB), >> binding to CREs near certain genes and acting as a transcription factor.

Many Eukaryotic mRNAs Are Subject to Translational Repression e.g., translational repressors (RNA-binding proteins) interact with initiation factors or with the ribosome to prevent or slow translation

Development Is Controlled by Cascades of Regulatory Proteins

Maternal Genes Christiane Nusslein-Volhard (p.1112) bicoid (bcd) gene product gradient Two posteriors

Segmentation genes e.g., ftz gene product (Ftz) Early embryo Late embryo

Homeotic genes: Antennapaedia (~ mouse HOX 1.1)

The discovry of structural determinants with identifiable molecular functions is the first step in understanding the molecular events underlying development. bithorax mutation