Control of gene expression in Eukaryotes

Influenced by chromosome organisation and chromatin modifications
1. during interphase of the cell cycle, chromosomes are unwound
2. chromosomal organization appears to be continuously rearranged so that transcriptionally active genes are cycled to the edge of chromosome at the border of the interchromosomal channels
3. when a gene has moved to the edge of a chromosome territory, chromatin structure must be remodelled, in order to make promoter sites accessible to the transcription machinery
Epigenetics
1. development and maintenance of an organism is orchestrated by a set of chemical reactions that switch parts of the genome off and on at strategic times and locations
2. Chromatin remodelling
  1. DNA in eukaryotic chromosomes is combined with histones and non histone proteins to form chromatin
  2. chromatin is characterised by the presence of repeating structures called nucleosomes
  3. nucleosomal DNA is further compacted in 30nm fibers and higher order structures and it inhibits many processes, including transcription, replication and DNA repair
  4. ability to alter the association of DNA with chromatin structures is essential to allow regulatory proteins to access DNA
3. Histone modifications
  1. histone acetyltransferase (HAT) lessens the attraction between histones and DNA
    1. HATs are targeted to genes by specific transcription factors
  2. the loosening of histones with DNA facilitates chromatin remodelling (opens chromatin and makes promoter regions available for binding to transcription factors that initiate the chain of events leading to gene transcription) catalysed by ATP-dependent chromatin remodelling complexes
  3. as well as acetylation, histones can be modified by phosphorylation and methylation which occur at specific amino acid residues in histones
4. DNA methylation
  1. adding or removing methyl groups on the bases in DNA
  2. an inverse relationship exists between the degree of methylation and the degree of gene expression
  3. methylation patterns are tissue specific and heritable for all cells in that tissue
  4. incorporation of 5-azacytidine causes undermethylation of sites and causes changes in the pattern of gene expression
  5. methylation may also directly repress transcription by interfering with the binding of transcription activators
Eukaryotic transcription
1. Regulated at cis-acting sites
  1. eukaryotic genes have several types of cis-regulatory sequences that control transcription (promoters, silencers and enhancers)
  2. because they function when adjacent to the structural genes they regulate
  3. transcription of DNA into an mRNA is a complex, highly regulated process involving several different types of DNA sequences, interactions of many proteins, chromatin remodelling and the bending and looping of DNA sequences
  4. expression of eukaryotic genes is controlled by regulatory elements directly adjacent to the gene, and by sequences that can be far from the transcriptional unit
  5. cis-acting regulatory sites influence transcription initiation by acting as binding sites for specific transcription regulatory proteins
  6. these transcription regulatory proteins, known as transcription factors, can have diverse and complicated effects on transcription
2. Promoters
  1. promoters are nucleotide sequences that serve as recognition sites for the transcription machinery and are necessary for transcription to be initiated at basal-level
  2. promoters are located immediately adjacent to the genes they regulate
  3. the core promoter consists of the TATA box (the region to which RNA polymerase II binds) and the start site, and CAAT boxes and GC boxes are elements that bind transcription factors
3. Enhancers
  1. they are position and orientation independent and can act at a distance to stimulate transcription initiation
  2. they are modular and often contain several short DNA sequences
  3. they are cis-regulators as they function when adjacent to the structural genes they regulate, as opposed to trans-regulators (e.g. binding proteins), which can regulate a gene on any chromosome
  4. enhancers are necessary for the full level of transcription
  5. enhancers are responsible for time- and tissue-specific gene expression
  6. enhancers are different from promoters as the position of an enhancer is not fixed, its orientation can be inverted without significant effect on its action and if an enhancer is experimentally moved to another location in the genome, or if an unrelated gene is placed near an enhancer, the transcription of the adjacent gene is enhanced
4. Silencers
  1. repress the level of transcription
  2. like enhancers, silencers are short DNA sequence elements, located in regions surrounding a promoter, that affect the rate of transcription
  3. they often act in tissue- or temporal-specific ways to control gene expression
5. Transcription factors
  1. proteins that bind to DNA and activate (or repress) transcription Initiation
  2. they have two functional domains (clusters of amino acids that carry out a specific function) -DNA binding domain and trans-activating or trans-repressing domain (bind to RNA polymerase or to other transcription factor at the promoter)
  3. domains take on several forms
    1. Helix-turn-helix (HTH)
      1. three planes of a-helix of the protein are established and these domains bind in the grooves of the DNA molecule
    2. Zinc finger (bZip)
      1. cysteine and histidine residues bind to a Zn++ atom and this loops the amino acid chain out into a finger like configuration that binds in the major groove of the DNA helix
      2. associated with growth, development and differentiation
    3. Basic leucine zipper (bleuZip)
      1. allows protein-protein dimerisation
      2. interactions of leucine residues at every other turn of the a-helix in facing stretches of two polypeptide chains and when the a-helical regions form a leucine zipper, the regions beyond the zipper form a Y-shaped region that grips the DNA in a scissor-like configuration
  4. will only become active when modified structurally (by phosphorylation or by binding to a co-activator such as a hormone)
  5. different transcription factors may compete for binding to a given DNA sequence or to two overlapping sequences
  6. multiple transcription factors that bind several enhancers and promoter elements within a gene regulatory region can interact with each other to fine-tune the levels and timing of transcription initiation
human metallothioneoin IIA (hMTIIA) gene
1. provides an example of how one gene can be transcriptionally regulated through the interplay of multiple promoter and enhancer elements and the transcription factors that bind to them
2. the glucocorticoid receptor (GCRc) protein binds to the GRE, but only when the receptor is bound to the glucocorticoid steroid hormone
3. hMTIIA gene has both basal and inducible cis-acting elements that bind different transcription factors
4. the product of the hMTIIA gene is a protein that binds to heavy metals, such as zinc and cadmium, which protects cells from the toxic effects of high levels of these metals
5. the gene is expressed at low levels in all cells but is transcriptionally induced to high levels when cells are in the presence of heavy metals and steroid hormones such as glucocorticoids
6. the GCRc is normally located in the cytoplasm of the cell however when glucocorticoid hormone enters the cytoplasm, it binds to the receptor and cause a conformational change that allows the receptor to enter the nucleus, bind to the GRE and stimulate MTIIA transcription
Post-transcriptional modifications
1. alternative splicing
  1. can generate different forms of mRNA from a pre-mRNA to give rise to a number of proteins from one gene
  2. group I introns (represented by introns that are part of the primary transcript of ribosomal RNAs) do not require any additional factors except guanosine for removal by splicing
  3. group II introns (present in the primary mRNA and tRNA) do not even require guanosine for self-excision
  4. as with group I splicing two transesterification reaction are involved: 1.the interaction of the 3’-OH group from and adenine (A) residue present within the branch point region of the intron and an intermediate structure is formed and 2.linking the cut 5’ end of the intron to the A resulting in the formation of the characteristic loop structure called a lariat, which contains the excised intron.
  5. other introns are spliced out by a spliceosome which consists of small nuclear RNA (snRNAs) complexed with proteins to form small nuclear ribonucleoproteins (snRNPs)
2. RNA editing
  1. the nucleotide sequence of the pre-mRNA is altered prior to translation
  2. Substitution editing
    1. occurs in mammals
  3. insertion/deletion editing
    1. in Trypanosomes, mitochondrial RNAs undergo extensive insertion/deletion editing and this editing is directed by guide RNA (gRNA) templates transcribed from the mitochondrial
Controlling mRNA stability
1. one way in which mRNA stability is controlled is through translational stability (a and b-tubulins)
2. the first four amino acids of the tubulin gene product constitute a recognition element to which regulatory factors (a and b-tubulins) bind
3. the interaction of the tubulin with the nascent protein activates an RNAse and it degrades the tubulin mRNA in the act of translation, shutting down tubulin biosynthesis
RNA silencing
1. first discovered in plants are short RNA molecules that regulate gene expression in the cytoplasm by repressing translation of mRNAs and degrading mRNAs
2. 1.Dicer has two catalytic domains (one domain in each monomer is inactive) and functions as a dimeric enzyme, and alignment and cutting by the active domain result in cleavage of the RNA 2.the product is a short RNA called short interference RNA (siRNA) which unwinds into sense and antisense single strands and the antisense strand combines with a protein complex, RISC (RNA-Induced Silencing Complex), that recognizes, binds to, and cleaves mRNA containing sequences complementary to the antisense strand of the siRNA 3.RISC cuts the mRNA at or near the middle of the region paired with the siRNA, and the mRNA fragments are degraded
3. a second form of RNA silencing is mediated by short RNA molecules called microRNAs (miRNAs)
  1. miRNA are short, single-stranded RNA about 20-25 nucleotides long, derived from longer (about 100 nucleotide) precursor RNAs that form hairpin structures
  2. these RNA precursors are cleaved by Dicer, and the resulting miRNAs pair with internal or 3’-untranslated regions (UTRs) of other mRNA molecules and this results in either a block to translation or the targeting of the mRNA for degradation
  3. small RNAs, processed by Dicer, play a role in RNA-directed DNA methylation (RdDM) and these RNAs combine with DNA methyl transferases (DMTases) to methylate cytosine residues in promoter regions
4. similar RNAs act in the nucleus to alter chromatin structure and bring about gene silencing
5. the best-studied form of RNA silencing is called RNA interference (RNAi) in animals and posttranscriptional gene silencing (PTGS) in plants where a gene-specific double-stranded RNA (about 70 nucleotides) is processed by a protein with double-stranded RNAse activity called Dicer

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Control of gene expression in Eukaryotes

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	Created by Abbey Roberts about 11 years ago