HSS2305C Fall 2024 Lecture 11 PDF

HSS2305: Molecular Mechanisms of Disease Lecture 11 – Control of Gene Expression – October 21, 2024 Section C00 Fall 2024 Dr. Alex Green  Nucleus Nucleus: Site of transcription Chromatin Ti...

HSS2305: Molecular Mechanisms of Disease Lecture 11 – Control of Gene Expression – October 21, 2024 Section C00 Fall 2024 Dr. Alex Green  Nucleus Nucleus: Site of transcription Chromatin Tightly packed DNA + Protein Complex Nucleolus Proteins for transcription transported into the nucleus RNA products for translation (mRNA, tRNA, rRNA) transported out of the nucleus Transport via the nuclear pores of the nuclear envelope Nuclear trafficking Nuclear Envelope Double membrane Outer membrane continuous with ER Inner membrane Lamina provides mechanical support and chromatin anchoring ~60 transmembrane proteins Ex. Nesprin 1/2 and Nesprin 3 for anchoring >1000 nuclear pore complexes Nuclear trafficking Nuclear Pore Complex (NPC) Nuclear pore complex Composed of Nucleoporins ~30 different Gateway across barrier for: RNAs Proteins Octagonal symmetry Diameter of central channel fluid (20-40 nm) Nucleoporins lining central channel have FG domain (phenylalanine-glycine repeats) Form hydrophobic mesh Block free diffusion of large macromolecules  Nuclear trafficking Transport of Proteins Across Nuclear Envelope For transport proteins contain specific AA sequences Nuclear Localization Signal (NLS) Nuclear Export Signal (NES) Transport receptors ferry proteins across envelope Importins heterodimeric receptor importin α/β binds to NLS Exportins and RanGTP bind to NES https://pubmed.ncbi.nlm.nih.gov/21487518/ Nuclear trafficking Transport of mRNA Across Nuclear Envelope: mRNAs Export mRNAs transported as ribonucleoproteins (RNPs) Associated proteins interact with FG domain of nucleoporins Only mature mRNAs (fully processed) are exported Nxf1 and Nxt1 Dependent Not exportin or RanGTP dependent https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008338 Genetic Blueprint and Transcriptional State Every cell in an organism: Same genetic information Same DNA Not every cell in an organism: Looks the same Acts the same Ex. myofibers vs skin fibroblasts Determined by genes that are expressed or “Transcriptional State” Tightly controlled Varies between cell types Varies under different conditions Not fixed Overview of gene regulation 1) 2) 3) 4) Overview of gene regulation 1) 1 - Transcriptional control Transcription Factors (TF) ~5-10% genome encodes transcription factors 1. General transcription factors Bind to core promoter sites, associate with RNA Pol TBP, TAF, TFIIA-H 2. Sequence specific transcription factors Bind to regulatory sites of particular gene Activators stimulate transcription Repressors inhibit transcription Each TF regulates many different genes Each gene regulated by many different TF Transcription Review https://youtu.be/JOBwqwxgJqc 1 - Transcriptional control Transcription Factors (TF) Basic Structures DNA-binding domain/motif Binds to specific sequences of base pairs on DNA Activation Activation domain domain Dimerization Site of interaction with domain other proteins Dimerization domain Promotes binding with DNA-binding another protein of domain identical or similar structure Dimer 2 protein structure 1 - Transcriptional control Transcription Factors (TF) DNA-binding motifs: Bind to DNA with Van der Waals forces ionic bonds hydrogen bonds Types: 1. Zinc fingers 2. Helix-Loop-Helix (HLH) 3. Leucine Zipper 1 - Transcriptional control Transcription Factors (TF) 1. Zinc fingers Zinc ion of each finger between: 2 cysteines (ß- sheet) 2 histidines (α-helix) Binds in major grooves in target DNA α-Helix = recognition domain Minor Groove Major Groove 1 - Transcriptional control Transcription Factors (TF) 2. Helix-Loop-Helix (HLH) 2 α-helical segments separated by loop Often preceded by stretch of highly basic AA Positive polar charged AA Charged side chains contact DNA DNA is negatively charged Often called basicHLH or bHLH Always occur in dimers hetero – 2 different proteins Homo – 2 same proteins 1 - Transcriptional control Transcription Factors (TF) 3. Leucine Zipper Series of α-helix chains Leucine (non-polar AA) every 7th AA Basic AA’s on helix recognize specific nucleotide sequence of DNA Completes the basic part of the Leucine Zipper Domain (basicZIP or bZIP) Exist as dimers Two α-helices interact together forming a ”zipper” or coiled coil 1 - Transcriptional control DNA sites for TF binding Where do Transcription Factors Bind? Response elements DNA site of transcription factor binding Response Elements 1 - Transcriptional control DNA sites Proximal and Distal Promoter Regulatory Sequences/Elements Type of response elements Bind transcription factors Regulate frequency of transcription Many have common consensus sequences for general transcription factors, ex: CAAT box (I.e. 5’-GGCCAATCT-3’) GC box (I.e. 5'-GGGCGG-3’) 1 - Transcriptional control DNA sites Enhancer sites Up to 50,000 base pairs upstream of transcription start site Transcription factors (activators) Bind enhancer regions Bind PIC or mediator Co-activators (mediator) Either: Binds PIC and transcription factor Converts chromatin to euchromatin (“loose chromatin”) Can alter level of transcription by 100+-fold  1 - Transcriptional control DNA sites Co-activators Protein that increases gene expression Does not directly bind DNA Therefore not a transcription factor! 2 Types: 1. Direct interactions with PIC 2. Interactions with histones to increase chromatin relaxation Upstream activating sequence  1 - Transcriptional control DNA sites Co-activators 1. Direct interactions with PIC Mediator complex that interacts with RNA Pol II of PIC includes a co-activator as a multi-subunit protein) Binds to an activator (i.e. a transcription factor) Essential to almost all protein-coding genes Upstream activating sequence 1 - Transcriptional control DNA sites Co-activators: 2. Interactions with Chromatin Remodels chromatin to make it regions of DNA more available for Histone transcription factor binding Also known as Chromatin Relaxation How? Coactivator Coactivator ATP hydrolysis to alter nucleosome structure and location 1. Promote sliding of histone along Coactivator DNA à new position 2. Conformational change of nucleosome Coactivator 3. Exchange for variant histone that promotes transcription 4. Displace histone from DNA completely  1 - Transcriptional control DNA sites In this example GR is the transcription factor Co-activators: 2. Interactions with Chromatin Histone acetyltransferases (HATs) Transfers acetyl groups to lysine residues of histones Acetyl group lowers positive charge of histones Reduces binding to negatively charged DNA Exposes binding site for chromatin remodelling complexes Ex. CBP (coactivator) Chromatin remodelling complexes Increases the accessibility of the promoter to transcription machinery Ex. SWI/SNF (coactivator) Both CBP and TFIID can acetylate and initiate transcription  1 - Transcriptional control DNA sites In this example GR is the transcription factor Co-activators: 2. Interactions with Chromatin Histone acetyltransferases (HATs) Transfers acetyl groups to lysine residues of histones Acetyl group lowers positive charge of histones Reduces binding to negatively charged DNA Exposes binding site for chromatin remodelling complexes Ex. CBP (coactivator) Chromatin remodelling complexes Increases the accessibility of the promoter to transcription machinery Ex. SWI/SNF (coactivator) Both CBP and TFIID can acetylate and initiate transcription  1 - Transcriptional control DNA sites In this example GR is the transcription factor Co-activators: 2. Interactions with Chromatin Histone acetyltransferases (HATs) Transfers acetyl groups to lysine residues of histones Acetyl group lowers positive charge of histones Reduces binding to negatively charged DNA Exposes binding site for chromatin remodelling complexes Ex. CBP (coactivator) Chromatin remodelling complexes Increases the accessibility of the promoter to transcription machinery Ex. SWI/SNF (coactivator) Both CBP and TFIID can acetylate and initiate transcription  1 - Transcriptional control DNA sites In this example GR is the transcription factor Co-activators: 2. Interactions with Chromatin Histone acetyltransferases (HATs) Transfers acetyl groups to lysine residues of histones Acetyl group lowers positive charge of histones Reduces binding to negatively charged DNA Exposes binding site for chromatin remodelling complexes Ex. CBP (coactivator) Chromatin remodelling complexes Increases the accessibility of the promoter to transcription machinery Ex. SWI/SNF (coactivator) Both CBP and TFIID can acetylate and initiate transcription Overview of gene regulation 2)  2 - mRNA Processing control RNA transcripts must undergo processing Only processed mRNA can exit the nucleus snRNPs Splicing via action of snRNPs occurs in nucleus before export Different splice sites can be repressed or activated Depends on nearby binding proteins SR proteins Activate splice sites Bind to exon/intron splicing enhancers (ESE/ISE) hnRNP (heterogeneous RNA+proteins) Repress splice sites Bind to exon/intron splicing silencers (ESS/IS) Alter binding localization of U2AF, U1 and U2 snRNPs to the transcript  2 - mRNA Processing control RNA transcripts must undergo processing Only processed mRNA can exit the nucleus snRNPs Splicing via action of snRNPs occurs in nucleus before export Different splice sites can be repressed or activated Depends on nearby binding proteins SR proteins Activate splice sites Bind to exon/intron splicing enhancers (ESE/ISE) hnRNP (heterogeneous RNA+proteins) Repress splice sites bind to exon/intron splicing silencers (ESS/IS) Alter binding localization of U2AF, U1 and U2 snRNPs to the transcript Overview of gene regulation 3)  3 - Translational control Once mRNA leaves the nucleus several regulatory mechanisms are present: 1. Initiation and progression of translation 2. Localization of mRNAs to certain sites within the cell 3. mRNA stability within the cytoplasm 5` and 3` untranslated region (UTRs) of mRNA play important role in regulation  3 - Translational control 1. Initiation and progression of *** ** translation GTP GDP Global regulation Affects translation of all mRNAs Phosphorylation of initiation factors inhibits translation Phosphorylation of eIF2 eIF2 cannot exchange GDP for GTP Initiating tRNA cannot bind to mRNA  3 - Translational control 1. Initiation and Heat shock progression of Viral infection Unfolded proteins *** ** translation AA starvation GTP GDP Global regulation Affects translation of P all mRNAs Phosphorylation of initiation factors inhibits translation Phosphorylation of eIF2 eIF2 cannot exchange GDP for GTP Initiating tRNA cannot bind to mRNA Occurs with stress  3 - Translational control 1. Initiation and Heat shock progression of Viral infection Unfolded proteins *** ** translation AA starvation GTP GDP Global regulation Affects translation of P all mRNAs Phosphorylation of initiation factors inhibits translation Phosphorylation of eIF2 eIF2 cannot exchange GDP for GTP Initiating tRNA cannot bind to mRNA Occurs with stress  3 - Translational control 1. Initiation and progression of translation Specific regulation Affects translation of specific mRNAs only UTR in mRNA have stem loops Proteins bind and regulate translation Ex. Ferritin Sequesters iron in cytoplasm of cell when iron levels are too high Ferritin mRNA translation is specifically regulated: Regulated by iron regulatory protein (IRP) Low [iron] à IRP binds iron- response element (IRE) in 5` UTR. Ribosome cannot bind to mRNA. High [iron] à IRP loses affinity for IRE causing dissociation and initiation of translation  3 - Translational control 2. Localization of mRNAs to certain sites within the cell Specific mRNAs are sequestered to functionally distinct cytoplasmic domains Ex. Polarized cells Allows for location specific protein expression Determined by localization sequence (“zip code”) in 3’ UTR RNA binding proteins bind and direct mRNA Microtubules transport the mRNA Microfilaments anchor mRNA During transport, bound proteins prevent translation  3 - Translational control 3. mRNA stability within the cytoplasm mRNA in cytoplasm continually translated into protein mRNA Half-lives range: 10 min -24 hrs Length of 3`poly(A) tail at the 3’UTR related to half life Start with ~ 200 adenosine residues Deadenylase (exonuclease) shortens 3’ Poly(A) tail in cytoplasm Leads to degradation Sequences in 3`UTR also determine poly(A) tail stability Binding sites for proteins that promote deadenylation Ex. AU-rich elements (AREs) destabilize mRNA Binding sites for microRNAs (miRNAs) that promote deadenylation  3 - Translational control 3. mRNA stability within the cytoplasm Unless protected mRNA with short (