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Regulation of Gene Expression

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Regulation of Gene Expression Regulation of Gene Expression Gene Expression Can be Regulated at Many of the Steps in the Pathway from DNA to RNA to Protein : (1) controlling when and how often a given gene is transcribed (2) controlling how an RNA transcript is spliced or otherwise processed (3) selecting which mRNAs are exported from the nucleus to the cytosol (4) selectively degrading certain mRNA molecules (5) selecting which mRNAs are translated by ribosomes (6) selectively activating or inactivating proteins after they have been made * most genes the main site of control is step 1: transcription of a DNA sequence into RNA. * Chromatin remodeling * controlling when and how often a given gene is transcribed ! DNA regulation ! Chromatin ! double helix accessibility ! gene and its surroundings ! Promoter/Operator (Bacteria) ! Promoter + enhancing region (Eukaryote ) ! Overview of Eukaryotic gene regulation Mechanisms similar to those found in bacteria-most genes controlled at the transcriptional level ! Gene regulation in eukaryotes is more complex than it is in prokaryotes because of: ! The larger amount of DNA ! Larger number of chromosomes ! Spatial separation of transcription and translation ! mRNA processing ! RNA stability ! Cellular differentiation in eukaryotes Transcription is the Most Regulated Step ! Transcription; from DNA to RNA, is catalyzed by the enzyme RNA polymerase. ! Initiation of transcription requires the formation of a complex between the promoter on the DNA and RNA polymerase. ! Initiation rate is largely controlled by the rate of formation of the complex DNA (promoter) - RNA polymerase. Rate = number of events per unit time. Transcriptional Control The Latin prefix cis translates to “on this side” “next to” ! cis-acting “next to” elements (cis-Regulatory Elements) (CREs) are regions of non-coding DNA which regulate the transcription of nearby genes ! trans-acting “across from” elements usually considered to be proteins, that bind to the cis-acting sequences to control gene expression. Trans- acting factors affect the expression of genes that may be physically located very far away, even on different chromosomes. The expression of a particular gene may be regulated by the concerted action of both cis and trans-acting elements 1) cis-acting elements: Promoter: is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites (TSS) of genes, on the same strand and upstream on the DNA (towards the 5 region) Promoters can be about 100–1000 base pairs long Promoter : Promoter elements: regulatory region upstream a gene ! Core promoter the minimum portion of the promoter required to initiate transcription includes: • The transcription start site (TSS) and elements directly upstream • A binding site for RNA polymerase • General transcription factor binding sites ! Proximal promoter the proximal sequence upstream of the gene that tends to contain primary regulatory elements, approximately 250 base pairs upstream of the start site ! Distal promoter (enhancers) the distal sequence upstream of the gene that may contain additional regulatory elements, often with a weaker influence than the proximal promoter The transcription start sites or START, is the first nucleotide at which transcription initiates, it means, it is at this site the first nucleotide is incorporated into the transcribed RNA. ! The start site is the first ntds and symbolized by +1, any sequence to the right of the start is called down stream elements and numbered as +10, +35 and so on. ! Any nucleotide present on the left of the start is denoted by (-) symbol and the region is called upstream element. The numbers are written as -10, -20, -35 etc. Bacterial promoters The promoter contains two short sequence elements approximately -10 and -35 nucleotides upstream from the transcription start site • The sequence at -10 (the -10 element) has the consensus sequence TATAAT • The sequence at -35 (the -35 element) has the consensus sequence TTGACA It should be noted that the above promoter sequences are recognized only by RNA polymerase holoenzyme containing sigma-70 The holoenzyme is for -10 sequence the complete complex T A T A A T containing all the 77% 76% 60% 61% 56% 82% subunits needed for activity. for -35 sequence T T G A C A 69% 79% 61% 56% 54% 54% • In prokaryotes there are other sequences in the upstream of the promoter, behind –35 sequences. Such sequences may present at • –65 to –60 or they may be present at –200 or they may be present at —1000 bp upstream or they may present in downstream regions. Consensus promoter sequence in E.coli: -35 TCTTGACAT—17+/- 1 — TATAAT-5-8 +1 A(G/T/C) RNA polymerase binding region- -35 --10 +1-----> TTGACA-------TATAAT------- +1----- Eukaryotic promoters 1) Gene promoters are typically located upstream of the gene and can have regulatory elements several kilobases away from the transcriptional start site (enhancers) • Located within 100 bp upstream contains TATA box consensus sequence TATAAA (25 – 30) bases upstream from start point, which is recognized by general transcription factor TATA-binding protein (TBP); and B recognition element (BRE), which is recognized by the general transcription The AT-rich sequence easy unwinding (because weaker base-stacking interactions among A and T rather than G and C). • Proximal promoter - the proximal sequence upstream of the gene that tends to contain primary regulatory elements, approximately 250 base pairs upstream of the start site ! CAAT box consensus sequence GGCCAATCT that occur upstream by 75-80 bases to the initial transcription site ! GC box consensus sequence GGGCGG that occur upstream by 110 bases upstream from the transcription initiation site Require the binding of several protein factors to initiate transcription (DNA binding domains on TFs – ‘motifs’) 2) Enhancers & Silencers far away from promoter Distal promoter - the distal sequence upstream of the gene that may contain additional regulatory elements, often with a weaker influence than the proximal promoter • They are called enhancer sequences. Here enhancer means it increases the efficiency of transcription by 100 to 200%. This is achieved through certain proteins bind to enhancer elements, and then contacts RNA holozyme by protein-protein interactions. Regulation Occurs at the Step of Initiation Regulation of Transcription The focal point is whether or not RNA polymerase binds the promoter of a gene and initiates transcription which depends on: 1) Affinity of RNA polymerase for a given promoter • some promoters are “strong” and bind RNA polymerase with high affinity • some promoters are “weak” and bind RNA polymerase with low affinity, requiring help from special proteins called transcription factors • the strength of a promoter depends on its sequence ! Two consensus sequences of nucleotides in the promoter region. A. a sequence at -10 (10 bases before the start site): TATAAT B. a sequence at -35: TTGACA 2 . Structural changes modifying the affinity of the RNA polymerase for the promoter. 3. Influence of proteins referred to as transcription factors, proteins can change the affinity of the RNA polymerase for the promoter. • Proteins that help RNA polymerase bind a promoter (referred to as “activators”) If the proteins increase the affinity, they are activators. • Proteins that inhibit or prevent RNA polymerase from binding a promoter (referred to as “repressors” or “inhibitors”) If the proteins decrease the affinity, they are repressors. Regulation of Gene Expression came originally from the study of how E. coli b a c t e r i a a d a p t t o changes in the Regulation of Gene Expression composition of their growth medium. Genes, based on their activity, can be grouped as housekeeping genes and others are classed as induced to express or express in stage specific or tissue specific way. ! Genes turned on all the time (Constitutive) Housekeeping genes express all the time under all normal conditions. ! Other genes can be regulated: – Turned On – Turned Off Regulation of gene expression allows cells to respond to changes in their environment 1- An inducible gene is a gene that is expressed in the presence of a substance (an inducer) in the environment. This substance can control the expression of one or more genes (structural genes) involved in the metabolism of that substance. For example, lactose induces the expression of the lac genes that are involved in lactose metabolism. An certain antibiotic may induce the expression of a gene that leads to resistance to that antibiotic. • Induction is common in metabolic pathways that result in the catabolism of a substance and the inducer is normally the substrate for the pathway. 2- Repressible gene is those in which the presence of a substance (a co- repressor) in the environment turns off the expression of those genes (structural genes) involved in the metabolism of that substance. e.g., Tryptophan represses the expression of the trp genes. ! Repression is common in metabolic pathways that result in the biosynthesis of a substance and the co-repressor is normally the end product of the pathway being regulated. Classification of enzymes by how gene expression is regulated ! Some enzymes are present at all times in a cell ! These are called constitutive enzymes ! The genes for constitutive enzymes are transcribed at a constant rate because they are always needed by cells ! RNA polymerase is a good example of a constitutive enzyme ! Some enzymes (Enzymes of a catabolic pathway) are needed only when a particular compound,
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