chapters 18 - regulation of gene expression in eukaryotes : levels of control of gene expression
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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 : - PowerPoint PPT PresentationTRANSCRIPT
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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)
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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.
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Eukaryote gene expression is regulated at six levels:
1. Transcription
2. RNA processing
3. mRNA transport
4. mRNA translation
5. mRNA degradation
6. Protein degradation
Fig. 18.1
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1. Transcription control of gene regulation is controlled by:
1. 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.
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1. Transcription control of gene regulation is controlled by:
2. 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).
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Fig. 18.2
Activation of transcriptionBy transcription factors (TFs), activator, and coactivator proteins.
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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.
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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.
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Fig. 18.10a
Chromatin remodelingAcetylation of histones enhances access to promoter region and facilitates transcription.
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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
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Fig. 18.13
Methylation of H19 inactivates transcription
(involved in expression of insulin like growth factor)
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Chromatic remodeling and DNA methylation are the basis for epigenetic inheritance.
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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.
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Galactose metabolizing pathway of yeast.
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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.
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Activation model of GAL genes in yeast.
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Fig. 18.4b
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Fig. 18.4c
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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.
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Examples of mammalian steroid hormones.
Steroid hormones are four ring structures/differences derive from differences in side-groups.
Plant hormones.
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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.
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Fig. 18.17, Model of glucocorticoid steroid hormone regulation.
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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
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Fig. 18.14, Alternative polyadenylation and splicing of the human CACL gene in thyroid and neuronal cells.
Calcitonin gene-related peptide
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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.
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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.
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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.
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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
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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
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Summary and contrasts:
Prokaryotes control expression by:
Transcription
Eukaryotes control expression by:
Transcription
RNA processing
mRNA transport
mRNA translation
mRNA degradation
Protein degradation
Fig. 18.1