Multiple levels of eukaryotic gene expression and regulation

Genomic ControlDNA Rearrangement: eg of gene regulationCassette Mechanism of the yeast mating-type switchChr 3 has 3 copies of mating type information. MAT locus contains alpha- or a- specific DNA and specifies mating type. HMRa and HML alpha: They are transcriptionally silenced by SIR genes.When alpha or a cell switches mating types the alpha or a DNA at the MAT locus is excised and a DNA cassette copy of alternating mating-type is inserted.Cells frequently switch mating type, as a means of maximizing opportunities for mating.

The yeast Saccharomyces cerevisiae is a simple single celled eukaryote with both a diploid and haploid mode of existence. The mating of yeast only occurs between haploids, which can be either the a or (alpha) mating type and thus display simple sexual differentiation. Mating type is determined by a single locus, MAT, which in turn governs the sexual behaviour of both haploid and diploid cells.

S. cerevisiae (yeast) can stably exist as either a diploid or a haploid. Both haploid and diploid yeast cells reproduce by mitosis, with daughter cells budding off of mother cells. Haploid cells are capable of mating with other haploid cells of the opposite mating type (an a cell can only mate with an cell, and vice versa) to produce a stable diploid cell. Diploid cells, usually upon facing stressful conditions such as nutrient depletion, can undergo meiosis to produce four haploid spores: two a spores and two spores.

Two haploid yeast of opposite mating types secrete pheromones, grow projections and mate.

a cells produce a-factor, a mating pheromone which signals the presence of an a cell to neighboring cells. Vice versa also.The different sets of transcriptional repression and activation which characterize a and cells are caused by the presence of one of two alleles of a locus called MAT: MATa or MAT. The alleles present at the MAT locus are sufficient to program the mating behaviour of the cell.

HML and HMR: the silent mating cassettesHaploid yeast switch mating type by replacing the information present at the MAT locus. For example, an a cell will switch to an cell by replacing the MATa allele with the MAT allele. This replacement of one allele of MAT for the other is possible because yeast cells carry an additional silenced copy of both the MATa and MAT alleles: the HML (Hidden MAT Left) locus typically carries a silenced copy of the MAT allele, and the HMR (Hidden MAT Right) locus typically carries a silenced copy of the MATa allele. The silent HML and HMR loci are often referred to as the silent mating cassettes, as the information present there is 'read into' the active MAT locus.

Location of the silent HML and HMR loci and the active MAT locus on yeast chromosome III.

Mechanics of the mating type switchThe process of mating type switching is a gene conversion event initiated by the HO gene. The HO gene is a tightly regulated haploid-specific gene that is only activated in haploid cells during the G1 phase of the cell cycle. The protein encoded by the HO gene is a DNA endonuclease, which physically cleaves DNA, but only at the MAT locus (due to the DNA sequence specificity of the HO endonuclease).

Once HO cuts the DNA at MAT, exonucleases are attracted to the cut DNA ends and begin to degrade the DNA on both sides of the cut site. This DNA degradation by exonucleases eliminates the DNA which encoded the MAT allele; however, the resulting gap in the DNA is repaired by copying in the genetic information present at either HML or HMR, filling in a new allele of either the MATa or MAT gene. Thus, the silenced alleles of MATa and MAT present at HML and HMR serve as a source of genetic information to repair the HO-induced DNA damage at the active MAT locus.

Figure 7-66 Molecular Biology of the Cell ( Garland Science 2008)

Genomic Control: Chromosome Decondensation and DNA accessibilityElaborate packaging of DNA with proteins to form euk. Chromosome adds a level of complexity.

To initiate transcription RNA pol must interact with DNA and a no. of specific proteins in promoter region.

Promoter embedded within highly folded and ordered chromosomal superstructure.

Some degree of chromatin decondensation (unfolding) is a critical event in euk. gene exp.

Visualization of Chromosome DecondensationVisual evidence for correlation between chromosome decondensation and transcription comes from studies on polytene chromosome of Drosophila.

Polytene chr. is a tightly attached pair of homologous chromosomes with a very large no. of precisely aligned, parallel chromatids. Visible in each of these chromosomes is a characteristic pattern of dark bands (highly condensed) and chromatin in a band is uncoiled during transcription.Transcriptional activity of polytene chromosomesTranscriptionally active regions light up under FM

Puffs in Polytene chromosomesActivation of genes of a given chromosome band causes coiled chromatin strands to unwind and expand outward resulting in a chromosome puff.Puffs are regions in which transcriptionally active region has become less condensed.

The puffing is direct visual manifestation of selective decondensation and transcription of specific segments of DNA

Nobel PrizesThe following scientists were recognized for their contributions to chromatin research with Nobel Prizes:

YearWhoAward1910Albrecht Kossel (University of Heidelberg)Nobel Prize in Physiology or Medicine "in recognition of the contributions to our knowledge of cell chemistry made through his work on proteins, including the nucleic substances"1933Thomas Hunt Morgan (California Institute of Technology)Nobel Prize in Physiology or Medicine "for his discoveries concerning the role played by the chromosome in heredity"1962Francis Crick, James Watson and Maurice Wilkins (MRC Laboratory of Molecular Biology, Harvard University and London University respectively)Nobel Prize in Physiology or Medicine "for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material"1982Aaron Klug (MRC Laboratory of Molecular Biology)Nobel Prize in Chemistry "for his development of crystallographic electron microscopy and his structural elucidation of biologically important nucleic acid-protein complexes"1993Roberts and SharpNobel Prize in Physiology "for their independent discoveries of split genes"2006Roger Kornberg (Stanford University)Nobel Prize in Chemistry "for his studies of the molecular basis of eukaryotic transcription"

Main control Point of gene regulation: Initiation of transcriptionA typical eukaryotic gene with its core promoter and proximal control regionA promoter is a DNA sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters are a means to demarcate which genes should be used for messenger RNA creation and thereby controls which proteins the cell manufactures.

Figure. Primary and secondary levels of gene regulation. According to this scheme, 'primary' regulation of genome expression occurs at the level of transcription initiation, this step determining which genes are expressed in a particular cell at a particular time and setting the relative rates of expression of those genes that are switched on. 'Secondary' regulation involves all steps in the gene expression pathway after transcription initiation, and serves to modulate the amount of protein that is synthesized or to change the nature of the protein in some way, for example by chemical modification.

Transcription in eukaryotic cellMore complex. Main differences:

Three diff RNA PolymerasesEuk. Promoters are more varied. 3 diff. types of promoters for 3 RNA polymerases. Great variation within each type especially one for protein coding genes.Many transcription factors are involved in binding of RNA Pol. to DNA.(Most of them must bind to DNA before Pol. can bind to promoter and initiate transcription. TFs determine specificity of transcription in eukaryotes).

Eukaryotic RNA PolymerasesStructurally all three are somewhat similar to each other and also to prokaryotic RNA pol.

Quite large with multiple polypeptide subunits and MW 500,000

Typical eukaryotic promoters used by RNA Polymerases I, II and IIIIIIIIItRNA gene5S rRNA genePromotersPol III is unusual (compared to Pol II) requiring no control sequences upstream of the gene, instead normally relying on internal control sequences - sequences within the transcribed section of the gene The process of transcription by Pol I is relatively unregulated (rRNA for ribosomes is always needed in large quantities). Consequently, transcription by Pol I is a comparatively simple process with few steps requiring regulation.

Transcription FactorsA transcription factor is a protein that binds DNA at a specific promoter or enhancer region or site, where it regulates transcription. Transcription factors can be selectively activated or deactivated by other proteins

Transcription factors possess several distinct functional domains:

DNA-binding domainTranscription regulation domain (in activators called as TAV)

Most TFs fall into categories based on structural motif present in DNA-binding domain.

There are three classes of transcription factors:

General transcription factors are involved in the formation of a preinitiation complex. The most common are abbreviated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH. They are ubiquitous and interact with the core promoter region surrounding the transcription start site(s) of all class II genes.

Upstream transcription factors are proteins that bind somewhere upstream of the initiation site to stimulate or repress transcription.

Inducible transcription factors are similar to upstream transcription factors but require activation or inhibition.

DNase I Sensitivity StudiesDemonstrates correlation between chromatin uncoiling and transcription.At low conc. DNase I preferentially degrades transcriptionally active DNA in chromatinDNA in condensed chromatin protected from DNaseI BRAINERYTHROCYTEGlobin gene is inactive in in brain cells: Present intact after nuclease treatmentGlobin gene is active in erythrocytes: DNA is vulnerable to nuclease attack. GlobinOvalbumin (control)

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