BSCI222 Exam III Lectures 17 22 Lecture 17 18 Gene Expression in Eukaryotes Chapter 17 2 7 1 17 18 14 16 18 16 8 9 18 1 4 7 8 11 1 16 8 20 22 26 29 o Differences from prokaryote regulation o o Nucleus o Large amounts of non coding DNA o Introns o Coupled prokaryote vs uncoupled transcription translation o Polycistronic prokaryote vs monocystronic o Lots of RNA modification o Multicellularity o Chromatin Modification of chromatin structure occurs in eukaryotes o DNase I Hypersensitivity o Several changes in chromatin are observed when genes become transcriptionally active o When they become transcriptionally active regions around the gene become highly sensitive to the action of DNase I o DNase I hypersensitive sites frequently develop about 1000 nucleotides upstream of the start site of transcription o Relaxation of chromatin allows regulatory proteins access to binding sites on the DNA o Histone modification o Two domains A globular domain that associates with other histones and the DNA A positively charged tail domain that probably interacts with the negatively charges phosphate groups on the backbone of the DNA o Tails are modified by the addition or removal of phosphate groups methyl groups or acetyl groups o Histone code because they encode information that affects how genes are expressed o Methylation of Histones o Addition of methyl groups to the tails of the histones o Addition can bring about the activation or repression of transcription o Addition of three methyl groups to lysine 4 in the tail of the H3 histone protein most common o Acetylation of Histones o Addition of acetyl groups usually stimulates transcription o Acetylation of histones controls flowering in Arabidopis FLC gene suppresses flowering until after an extended period of coldness FLD stimulates flowering by repressing the action of FLC FLD encodes a deacetylase enzyme which removes acetyl groups from histone proteins in the chromatin surrounding FLC The removal of acetyl groups from histones alters chromatin structure and inhibits transcription o Chromatin Remodeling o Chromatin remodeling complexes Some transcription factors and other regulatory proteins alter chromatin structure without altering the chemical structure of histones directly SWI SNF found in yeast o Major modes of Regulation o Transcriptional control o Post transcriptional control o Translational control o Transcriptional control o Initiation Access to promoters controlling chromatin chromatin modification Rate of assembly of initiation RNA Pol II Pre initiation complex gun Rate of assembly can vary Activator proteins Transcription factors o Stimulate and stabilize the basal transcription apparatus at the core promoter o Mediator transcriptional activator proteins make contact with the mediator and affect the rate at which transcription is initiated o Two functions 1 capable of binding DNA at a specific base sequence usually a consensus sequence in a regulatory promoter or enhancer and 2 able to interact with other components of the transcriptional apparatus and influence the rate of transcription o Bind sequences in promoters distal enhancers o Recruit components to promoter Rate of transition to elongation Activators interact with mediator Mediator interacts with Pol II Mediator is like a switchboard A mediator is a multi protein complex Integrates signals that affect Pol II Carboxy terminal domain of Pol II is phosphorylated does this trigger the switch to elongation Consists of tandem repeats of YSPTSPS How to prevent shorts activators interacting with the wrong intitiation complex INSULATORS Most enhancers are capable of stimulating any promoter in their vicinities Their effects are limited however by insulators which are DNA sequences that block or insulate the effect of enhancers in a position dependent manner Genetic insulators prevent activators from interacting with the wrong o Post Transcriptional regulation alternative splicing T antigen gene SF2 enhances the production of mRNA encoding the small t SF2 has two binding domains one domain is an RNA binding region and the other has alternating serine and arginine amino acids Splicing can be regulated by splicing activators and antigen repressors Example from slideshow exon 4 has weak 3 splice site too weak and splicing doesn t occur In females the Tra protein is expressed and acts as a splicing activator 3 5 and 3 4 5 o Translational control global Phosphorylating elF2 inactivates it all translation stops Mechanisms of gene regulation by RNA interference Small interfering RNAs and microRNAs regulate gene expression through at least 4 distinct mechanisms 1 cleavage of mRNA 2 inhibition of translation 3 transcriptional silencing 4 degradation of mRNA RNA cleavage o RISCs that contain an siRNA pair with mRNA molecules and cleave the mRNA near the middle of the bound siRNA o Cleavage is sometimes referred to as slicer activity o After cleavage the mRNA is further degraded Inhibition of translation o Some miRNAs regulate genes by inhibiting the translation of their complementary mRNAs o The exact mechanism by which mrRNAs repress translation is not known Transcriptional silencing o Other siRNAs silence transcription by altering chromatin structure o siRNAs combine with proteins to form a complex called RITs which is analogous to RISC o The siRNA component of RITS then binds to its complementary sequence in DNA or an RNA molecule in the process of being transcribed and represses transcription by attracting enzymes that methylate the tails of histone proteins Slicer independent degradation of mRNA o A final mechanism by which miRNAs regulate gene expression is by triggering the decay of mRNA in a process that does not require slicer activity Some genes are regulated by processes that affect translation or by modifications of proteins Epigenetics Imprinting same genotype different phenotype Heritable changes in gene expression not associated with changes in the DNA code Human example o If deletion is inherited from father prader willi syndrome mother angelman syndrome Same genotypes different phenotypes Mechanisms o DNA methylation Usually occurs at CpGs methyltransferase Because it occurs at CpGs patterns can be copied following replication So methylation patterns can be inherited Methylated DNA is transcriptionally inactive o Chromatin modification Many diseases are epigenetic in origin things we do and experience can impact gene expression in future generations Mechanism of dsRNA