Chapters 18 - Regulation of gene expression in eukaryotes: Levels of control of gene expression Short term control (to meet the daily needs of the organism) Long term control (gene regulation in development/differentiation)

Differences between prokaryotes and eukaryotes: Prokaryote gene expression typically is regulated by an operon, the collection of controlling sites adjacent to polycistronic protein-coding sequences. Eukaryotic genes also are regulated in units of protein-coding sequences and adjacent controlling sites, but operons are not known to occur. Eukaryotic gene regulation is more complex because eukaryotes possess a nucleus. (transcription and translation are not coupled). Two “categories” of eukaryotic gene regulation exist: Short-term - genes are quickly turned on or off in response to the environment and demands of the cell. Long-term - genes for development and differentiation.

Eukaryote gene expression is regulated at six levels: Transcription RNA processing mRNA transport mRNA translation mRNA degradation Protein degradation Fig. 18.1

1. Transcription control of gene regulation is controlled by: Promoters Occur upstream of the transcription start site. Some determine where transcription begins (e.g., TATA), whereas others determine if transcription begins. Promoters are activated by specialized transcription factor (TF) proteins (specific TFs bind specific promoters). One or many promoters (each with specific TF proteins) may occur for any given gene. Promoters may be positively or negatively regulated.

1. Transcription control of gene regulation is controlled by: Enhancers Occur upstream or downstream of the transcription start site. Regulatory proteins bind specific enhancer sequences; binding is determined by the DNA sequence. Loops may form in DNA bound to TFs and make contact with upstream enhancer elements. Interactions of regulatory proteins determine if transcription is activated or repressed (positively or negatively regulated).

Fig. 18.2 Activation of transcription By transcription factors (TFs), activator, and coactivator proteins.

More about promoters and enhancers: Some regulatory proteins are common in all cell types, others are specific. Each promoter and enhancer possesses a specific set of proteins (coactivators) that determines expression. Rate of gene expression is controlled by interaction between positive and negative regulatory proteins. Combinatorial gene regulation; enhancers and promoters bind many of the same regulatory proteins, implying lots of interaction with fine and coarse levels of control.

Chromosome structure, eukaryote chromosomes are packed with histones: Prokaryotes lack histones and other structural proteins, so access to the DNA is straightforward. Eukaryotes possess histones, and histones repress transcription because they interfere with proteins that bind to DNA. Verified by DNase I sensitivity experiments: DNase I readily degrades transcriptionally active DNA. Histones shield non-transcribed DNA from DNase I, and DNA does not degrade as readily. If you experimentally add histones and promoter binding proteins; histones competitively bind to promoters and inhibit transcription. Transcriptionally active genes possess looser chromosome structures than inactive genes. Histones are acetylated and phosphorylated, altering their ability to bind to DNA. Enhancer binding proteins competitively block histones if they are added experimentally with histones and promoter-binding TFs. RNA polymerase and TFs “step-around” the histones/nucleosomes and transcription occurs.

Fig. 18.10a Chromatin remodeling Acetylation of histones enhances access to promoter region and facilitates transcription.

DNA methylation and transcription control: Small percentages of newly synthesized DNAs (~3% in mammals) are chemically modified by methylation. Methylation occurs most often in symmetrical CG sequences. Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes. A gene for methylation is essential for development in mice (turning off a gene also can be important). Methylation results in a human disease called fragile X syndrome; FMR-1 gene is silenced by methylation. Fig. 18.12

Fig. 18.13 Methylation of H19 inactivates transcription (involved in expression of insulin like growth factor)

Chromatic remodeling and DNA methylation are the basis for epigenetic inheritance.

Short-term - transcriptional control of galactose-utilizing genes in yeast: 3 genes (GAL1, GAL7, & GAL 10) code enzymes that function in the galactose metabolic pathway. GAL1 galactokinase GAL7 galactose transferase GAL10 galactose epimerase Pathway produces d-glucose 6-phosphate, which enters the glycolytic pathway and is metabolized by genes that are continuously transcribed. In absence of galactose, GAL genes are not transcribed. GAL genes rapidly induced by galactose and absence of glucose. Analagous to E. coli lac operon repression by glucose.

Galactose metabolizing pathway of yeast.

Short-term - transcriptional control of galactose-utilizing genes in yeast: GAL genes are near each other but do not constitute an operon. Additional unlinked gene, GAL4, codes a repressor protein that binds a promoter element called an upstream activator sequence (UASG). UASG is located between GAL1 and GAL10. Transcription occurs in both directions from UASG. When galactose is absent, the GAL4 product (GAL4p) and another product (GAL80p) bind the UASG sequence; transcription does not occur. When galactose is added, a galactose metabolite binds GAL80p and GAL4p amino acids are phosphorylated. Galactose acts as an inducer by causing a conformation change in GAL4p/GAL80p.

Activation model of GAL genes in yeast.

Fig. 18.4b

Fig. 18.4c

Hormone regulation - another good example of short-term regulation of transcription: Cells of higher eukaryotes are specialized and generally shielded from rapid changes in the external environment. Hormone signals are one mechanism for regulating transcription in response to demands of the environment. Hormones act as inducers produced by one cell and cause a physiological response in another cell. Hormones act only on target cells with hormone specific receptors, and levels of hormones are maintained by feedback pathways. Hormones deliver signals in two different ways: Steroid hormones pass through the cell membrane and bind cytoplasmic receptors, which together bind directly to DNA and regulate gene expression. Polypeptide hormones bind at the cell surface and activate transmembrane enzymes to produce second messengers (such as cAMP) that activate gene transcription.

Examples of mammalian steroid hormones. Steroid hormones are four ring structures/differences derive from differences in side-groups. Plant hormones.

Hormone regulation continued: Genes regulated by steroid hormones possess binding regions in the sequence called steroid hormone response elements (HREs). HREs often occur in multiple copies in enhancer sequence regions. When steroid is absent: receptor is bound and “guarded” by chaperone proteins; transcription does not occur. When steroid is present: Steroid displaces the chaperone protein, binds the receptor, and binds the HRE sequence; transcription begins.

Fig. 18.17, Model of glucocorticoid steroid hormone regulation.

2. RNA processing control: RNA processing regulates mRNA production from precursor RNAs. Two independent regulatory mechanisms occur: Alternative polyadenylation = where the polyA tail is added Alternative splicing = which exons are spliced Alternative polyadenylation and splicing can occur together. Examples: Human calcitonin (CALC) gene in thyroid and neuronal cells Sex determination in Drosophila

Fig. 18.14, Alternative polyadenylation and splicing of the human CACL gene in thyroid and neuronal cells. Calcitonin gene-related peptide

Fig. 19.13 Alternative splicing in sex determination of Drosophila Sex is determined by X:A ratio. Sxl (sex lethal) gene determines the pathways for males and females. If X:A = 1, all introns and exon 3 (which contains the stop codon) are removed. If X:A = 0.5, no functional protein is produced.

3. mRNA transport control: Eukaryote mRNA transport is regulated. Some experiments show ~1/2 of primary transcripts never leave the nucleus and are degraded. Mature mRNAs exit through the nuclear pores.

4. mRNA translation control: Unfertilized eggs are an example, in which mRNAs (stored in the egg/no new mRNA synthesis) show increased translation after fertilization). Stored mRNAs are protected by proteins that inhibit translation. Poly(A) tails promote translation. Stored mRNAs usually have short poly(A) tails (15-90 As vs 100-300 As). Specific mRNAs are marked for deadenylation (“tail-chopping”) prior to storage by AU-rich sequences in 3’-UTR. Activation occurs when an enzyme recognizes AU-rich element and adds ~150 As to create a full length poly(A) tail.

5. mRNA degradation control: All RNAs in the cytoplasm are subject to degradation. tRNAs and rRNAs usually are very stable; mRNAs vary considerably (minutes to months). Stability may change in response to regulatory signals and is thought to be a major regulatory control point. Various sequences and processes affect mRNA half-life: AU-rich elements Secondary structure Deadenylation enzymes remove As from poly(A) tail 5’ de-capping Internal cleavage of mRNA and fragment degradation

6. Post-translational control - protein degradation: Proteins can be short-lived (e.g., steroid receptors) or long-lived (e.g., lens proteins in your eyes). Protein degradation in eukaryotes requires a protein co-factor called ubiquitin. Ubiquitin binds to proteins and identifies them for degradation by proteolytic enzymes. Amino acid at the N-terminus is correlated with protein stability and determines rate of ubiquitin binding. Arg, Lys, Phe, Leu, Trp 1/2 life ≤3 minutes Cys, Ala, Ser, Thr, Gly, Val, Pro, Met 1/2 life ≥ 20 hours

Summary and contrasts: Prokaryotes control expression by: Transcription Eukaryotes control expression by: RNA processing mRNA transport mRNA translation mRNA degradation Protein degradation Fig. 18.1