BY450 Genetic Control Mechanisms 1 Updated SC PDF

BY450 Genetic Control Mechanisms 1: Controlling Gene expression Dr Nigel Brissett Controlling Gene Expression Dr Nigel Brissett gene expression -control at multiple steps  gene expression -control at multiple steps Essential Cell Biology 3/e 2010 ...

BY450 Genetic Control Mechanisms 1: Controlling Gene expression Dr Nigel Brissett Controlling Gene Expression Dr Nigel Brissett gene expression -control at multiple steps  gene expression -control at multiple steps Essential Cell Biology 3/e 2010  Transcriptional control- bacterial operons operon - a functioning unit of genomic DNA containing a cluster of genes under the control of a single promoter p o Gene 1 Gene 2 Gene 3 Promoter Operator Gene(s) Regulatory gene  E. coli lac operon- genes b-galactosidase lactose permease transacetylase control of gene expression for efficient substrate use presence of lactose induces synthesis of enzymes for its metabolism (E.coli)  lac operon: expression inhibited by repressor protein i gene encodes repressor protein which binds to operator site on b-galactosidase (z) permease (y); lac operon and blocks transacetylase (a) transcription of z, y, a repressor genes  lac operon: expression inhibited by repressor protein Allolactose (derived from lactose) binds to repressor protein and blocks its action- transcription proceeds lac operon: example of an inducible expression system also- repressible systems eg trp operon codes for genes responsible for synthesis of amino acid tryptophan  trp operon Gene expression is decreased in the presence of a repressor e.g. the product of a biosynthetic pathway (Tryptophan) X Structural genes E, D, C, B, A: tryptophan synthase r p o E D C B A low tryptophan levels- repressor inactive, operon active expressed, repressor X tryptophan synthesised Tryptophan synthase repressor trp protein high tryptophan levels- trp binds to repressor, operon expression (inactive) inhibited trp  Transcriptional control in eukaryotes In eukaryotes, transcription is chiefly controlled by regulatory transcription factors* (TFs) TFs which activate or inhibit transcription are called transcriptional activators or repressors/silencers Operate through complex mechanisms, including chromatin modification *cf general transcription factors covered earlier eg TfIID involved with transcription initiation Action of transcriptional activators/repressors Interaction of regulatory TFs with Mediator and general Transcription Factors Activation domain interacts with Mediator or other components of transcriptional machinery transcription initiation complex (see previous lecture) DNA binding domain recognises a specific DNA sequence Multiple TFs may work together to control gene expression of a particular gene Transcription factors can bind at distant regulatory regions of DNA Many TFs exert influence by interacting with co-activators or co-repressors These include chromatin remodeling complexes and histone-modifying enzymes Histones and chromatin structure Chromatin remodeling and transcriptional activity changes in the puffing pattern of equivalent segments of chromosome 3 in Drosophia melanogaster over the course of around 20 hours of normal development. The more open the conformation of chromatin, the more transcriptional activity- euchromatin Highly condensed chromatin is normally transcriptionally silent- heterochromatin  Histone proteins chromatin (2x) H2A, H2B, H3, H4 (nucleosome) structure plus histone H1 (‘linker DNA’ associated) Chromatin alteration- role of histone modifying enzymes Enzyme-catalysed histone modification eg: histone acetyltransferases (HATs) histone deacetylases (HDACs) histone methyltransferase histone demethylation  Control of chromatin condensation by histone modification (grey) Figure 5-23 Essential Cell Biology (© Garland Science 2010) Acetylation or methylation of specific amino acids on specific This can alter the way in which histone proteins can change the histones of neighboring nucleosomes charge of the protein interact with one another  Correlation between transcriptional activity and histone modification Acetylation tends to increase (A) transcriptional activity. Acetylation of lysine 16 on H4 interferes with the formation of the 30-nm chromatin fibre, keeping chromatin in a highly opened state. Reversed by Deacetylation process. Methylation can cause transcriptional activation (A) or repression (R) depending on the residue. Heterochromatin is rich in methylated H3 residues DNA can be modified too…  Transcriptional repression by DNA methylation DNA Methylation is carried out by DNA methyl- transferases Main targets are cytosines in CpG dinucleotides throughout the genome Promoter methylation results in transcriptional silencing Methylated DNA is thought to recruit co-repressors eg. histone deacetylases Mammalian methyl-cytosine residues are part of a 5’-CpG-3’ dinucleotide within a symmetrical sequence  alternative splicing gene regulation… have covered transcriptional control so far several other important sites one final point covered here  RNA processing control – alternative splicing Genes can be alternatively spliced, and so encode more than one protein Gene containing exons A B C A B C A B C A B Alternative mRNA transcripts A C B C RNA processing control – alternative splicing tropomyosin regulation Translation- overview Translation initiation- components amino acyl tRNA ribsome large unit mRNA ribosome small unit Translation is mediated by a complex set of translation factors Role Prokaryotes Eukaryotes Initiation IF1 eIF1, eIF1A, eIF2, eIF2B, IF2 eIF3, eIF4A, eIF4B, IF3 eIF4E, eIF4G, eIF4H, eIF5, eIF5B Elongation EF-Tu, eEF1a EF-Ts eEF1bg EF-G eEF2 Termination RF-1, RF-2, RF-3 eRF1, eRF3 Cooper (2016), Table 9.1  Translation initiation in eukaryotes requires mRNA, amino-acyl tRNA (met), ribosome, GTP, 12 protein initiation factors 1. translocation of 40S ribosome initiation complex along mRNA …… AUG identified  Translation initiation in eukaryotes 2. release of translation factors 3. 60S subunit joins 40S/tRNA complex 4. 80S ribosome- met tRNA initiation complex completed  Translation initiation in eukaryotes requires mRNA, amino-acyl tRNA (met), ribosome, GTP, 12 protein initiation factors Regulation of translation initiation repressor proteins agents micro RNAs specific mRNAs action global translational activity  Regulation of translation initiation Example 1. Repressor binding to 5’ untranslated sequences Ferritin (iron-storage protein) repressor proteins agents micro RNAs specific mRNAs action global translational activity Regulation of ferritin translation by Iron Regulatory Protein (IRP) IRE: iron-response element in mRNA 5’ region adequate iron levels- normal translation proceeds Regulation of ferritin translation by Iron Regulatory Protein (IRP) Low iron levels: IRP binds to iron-response element (IRE)- interferes with translation initiation Translation halts  Regulation of translation initiation repressor proteins agents micro RNAs specific mRNAs action global translational activity  Regulation of translation initiation repressor proteins agents micro RNAs specific mRNAs action global translational activity Translational control by miRNA Hairpin structures form in primary single stranded miRNA transcripts a. miRNA duplex formation Translational control by miRNA Hairpin structures form in primary single stranded miRNA transcripts 1. miRNA duplex formation Sequential cleavage by Drosha and Dicer endonucleases. Yields double stranded miRNA Approx. 22 nucleotides b. miRNA interacts with RISC complex- strands unwind c. ss miRNA interacts with 3’ UTR of target mRNA Translational inhibition: directly by eIF4A/translation initiation interference; indirectly by deadenylation and increased mRNA degradation miRNAs influence many cell processes including cell cycle apoptosis cell differentiation  Regulation of translation initiation repressor proteins agents micro RNAs specific mRNAs action global translational activity  Regulation of translation initiation repressor proteins (protein kinases + agents translation initiation factors) micro RNAs specific mRNAs action global translational activity Regulation of translation by phosphorylation of eIF2 and eIF2b 1. Active eIF2-GTP escorts initiator Met-tRNA to ribosome. Inactive eIF2-GDP is released. 2. Active eIF2-GTP regenerated by action of eIF2B Regulation of translation by phosphorylation of eIF2 and eIF2b 2. Blocks reactivation of GDP-eIF2 to GTP- 1. Regulatory protein kinase enzymes eIF2 phosphorylate eIF2 or eIF2B  Regulation of translation initiation repressor proteins (protein kinases + agents eIF binding proteins) micro RNAs specific mRNAs action global translational activity Regulation of translation initiation by eIF4E- binding proteins (4E-BPs) in absence of growth factors, translation initiation blocked by 4E-BPs Regulation of translation initiation by eIF4E- binding proteins (4E-BPs) Growth factors stimulate phosphorylation of 4E-BPs which dissociate from eIF4E-Translation proceeds gene expression -control at multiple steps and finally………………. Essential Cell Biology 3/e 2010 mRNA degradation mRNA degradation is usually initiated by deadenylation of the poly-A tail followed by 3’ to 5’ exonuclease digestion mRNA degradation Also, 5’ to 3’ exonuclease digestion follows removal of 5’ meG ‘cap’  Regulation of gene expression via mRNA degradation In prokaryotes, most mRNAs have short half life (minutes) In eukaryotes, different mRNAs are degraded at different rates and can be a point of control of gene expression. structural proteins regulatory proteins 30min 20hrs core metabolic eg transcription factors enzymes Control by: miRNAs (deadenylation, see above) RNA-binding proteins RNA-binding proteins influence mRNA fate by binding to regulatory sequences RBPs Rapidly degraded RNAs often contain specific AU-rich sequences near 3’ ends (AREs) AREs act as binding sites for RNA-binding proteins (RBPs) that stabilise or target mRNA for degradation. Activities of RBPs are regulated by extracellular signals (eg. growth factors, hormones). RBPs control other key RNA processes – splicing, maturation, transport, translation  Summary Regulation of: a) Translation b) mRNA degradation a) Translation regulation agent Action on mechanism example Repressor protein specific mRNAs Binding to mRNA 5’UTR; Ferritin mRNA blocks initiation Iron response protein Repressor protein specific mRNAs Binding to mRNA 3’ UTR; neuroguidin blocks initiation miRNA specific mRNAs Binding to mRNA 3’ UTR; P53 tumour suppressor blocks initiation Repressor protein Global translation Kinase inhibition; initiation Growth factors facilitated action via eIF2, eIF2b Repressor protein Global translation Kinase activation; initiation Growth factors facilitated action via 4E-BP/eIF4E  b) RNA degradation process Regulation 5’ exonuclease Removal of 5’ MeG cap 3’ exonuclease Poly A tail removal; enhanced by miRNA/RISC interaction with 3’UTR of mRNA 3’ exonuclease RBP binding to AREs in 3’ UTR  Summary Prokaryotes can control the synthesis of enzymes in response to substrate/product levels using inducible or repressible operons Eukaryotic DNA is organised into chromatin that can be condensed or open/transcriptionally active or inactive Gene expression in eukaryotes is chiefly controlled by transcription factors which interact with other enzymes (HDACs, HATs, chromatin remodelling complexes) to activate or repress transcription Other mechanisms also play a role in controlling gene expression e.g. alternative splicing  Key texts The following are all good texts that cover the same material. Most give you MUCH more detail: – Cooper, G.M. & Hausman, R.E. (2019). The cell, a molecular approach (8th ed.). Blackwells. http://www.ncbi.nlm.nih.gov/books/NBK9839/ (2nd Ed) – Lodish et al. (2012). Molecular Cell Biology, 7th Ed, Freeman; https://www.ncbi.nlm.nih.gov/books/NBK21475/ (4th Ed) – Alberts et al. (2015). Molecular Biology of the Cell (6th ed). Garland Science. http://www.ncbi.nlm.nih.gov/books/NBK21054/ (4th Ed)  Online resources Other animations and video tutorials DNA learning centre – Cold Spring Harbours online DNA learning resource - http://www.dnalc.org/ DNA from the beginning – An animated primer of 75 experiments that made modern genetics http://www.dnaftb.org/ Bozeman Science basic video tutorial, Transcription and translation http://youtu.be/h3b9ArupXZg