Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35MCB 250 Exam IIIWednesday November 12th 7:00-9:00 PMSection Teaching Assistant RoomADQ, ADR, ADS, ADT, ADU, ADX, ADY, ADZSunetra Biswas, Preeti Ragunathan141 Loomis LaboratoryADE, ADF, ADG, ADH William Arnold 151 Loomis LaboratoryADA, ADB, ADC, ADV, ADM, ADOXinyun Cao, Michael Tencati114 David Kinely HallADI, ADJ, ADK, ADL, ADNTulip Mahaseth, Koh-Eun Narm100 Materials Science and Engineering (MSEB)Gene regulation in eukaryotes, Part ICore promoterTranscription unit (5' 3')5' enhancers3' enhancersLike prokaryotic genes, each eukaryotic transcription unit has a core promoter associated with its +1 transcription start site. [Note: a few genes have 2 or more core promoters that can serve as alternative sites of transcription initiation.]Organization of protein-coding genes in eukaryotesUnlike prokaryotes, in eukaryotes RNA polymerase is bound to the DNA indirectly by a group of proteins known as general transcription factors.Once these transcription factors have assembled on the promoter, they recruit RNA Polymerase to the DNA. Together, they are often referred to as the ‘general transcription machinery’.+1Watson, Fig. 13-16Core promoterTranscription unit (5' 3')5' enhancers3' enhancersIn addition to its core promoter, eukaryotic promoters contain a large number of cis-regulatory elements, generically known as enhancers.•Enhancers vary greatly in number from one gene to the next.•Enhancers are dispersed along the chromosome: they can be tens or hundreds of kilobases from the core promoter. •The majority of enhancers are located upstream of the core promoter, i.e. 5' to the transcription unit. Some genes also have enhancers within or 3' to the transcription unit.Organization of protein-coding genes in eukaryotesEnhancers influence transcription by binding sequence-specific regulatory proteins known as (specific) transcription factors. [Note: The term “specific” is usually implied rather than stated.]The transcription factors that bind to enhancers differ from the general transcription factors in several important regards.General TF’s•Small number of proteins (≈30). •Expressed in essentially all cells, and required for essentially all transcription. E.g. Pol II and its general TFs transcribe all protein-coding mRNAs.(Specific) TF’s•Large and diverse class of proteins [>2,000 in humans].•Some activate transcription; some repress; some do both.•Not required for transcription per se.•Most specific TF proteins are only expressed in a subset of cells, and within those cells they only influence a subset of genes.Differential gene expression in animalsExpression of peptide hormones in human pancreas.Feather specification in a chicken embryo.A map of transcription factor binding sites in the promoter of the sea urchin gene endo-16. Yu et al. (2001). Development 128, 617-629•The entire endo-16 regulatory region is small: 2.3 kb in length.•Within this DNA researchers have identified 34 distinct enhancer sequences (red boxes) that can bind proteins which regulate endo-16 transcription.•In total there are 16 different transcription factors (colored balloons) that - by binding to one or another of these enhancers - can influence the rate of endo-16 transcription.A single eukaryotic enhancer often binds multiple transcription factors which interact together to control transcription.E.g. the immune system activates transcription of the B-interferon gene in response to viral infections. This activation is largely controlled by a single enhancer element with 4 closely spaced transcription factor binding sites.Watson, Fig. 19-19Watson, Fig. 19-19Four different transcription factor proteins bound to the b-interferon enhancer.DNADNAThere are several major classes of eukaryotic transcription factors, which are classified by the structure of their DNA-binding domains:•Homeodomain proteins - The homeodomain evolved from the 'helix-turn-helix' DNA binding motif prevalent in bacteria. But unlike the bacterial proteins, homeodomains can bind DNA as monomers or dimers.•Basic helix-loop-helix (bHLH) proteins - Distinct from helix-turn-helix. Only bind DNA as dimers.•Basic leucine zipper (bZIP) proteins - The backbone of a bZIP protein is a dimerized leucine zipper. Each subunit has a basic a-helix that it inserts into the major groove of DNA. •Zinc finger proteins - Zinc finger proteins require Zn+2 ions to maintain their 3-dimensional conformation, and use a so-called ‘finger domain' to read the major groove. Can bind DNA as monomers or dimers.Watson, Fig. 19-5HomeodomainLike the bacterial helix-turn-helix motif, a homeodomain has a recognition helix (3) that reads the DNA sequence in the major groove and is stabilized by a second a-helix (2) …… but also has a third a-helix (1) that reads two base pairs in the minor groove.Many eukaryotic transcription factors can bind DNA as either homo- or heterodimers. Heterodimerization can alter sequence specificity of DNA binding, effectively reprograming the protein to bind the enhancers of a different set of target genes.bZIP transcription factorSubunit 1Subunit 2DNAATF/ATF homodimerATF/Jun heterodimer5'-TGACGTCA-3'3'-ACTGCAGT-5'5'-TGACATAG-3'3'-ACTGTATC-5'ATFATFATFJunOne important complication in the transcription of eukaryotic genes is the dense packaging of DNA into nucleosomes.Hence, eukaryotic genes that are not being actively transcribed are in a default state of transcriptional repression.Chromatin structure has important implications for the transcription potential of the DNA.Little or no transcriptionActive transcriptionLow level transcriptionWatson, Fig. 8-32Heterochromatin EuchromatinGRADED EFFECTIn this example, an activating transcription factor binds to enhancer DNA upstream of a promoter blocked by a nucleosome.Once bound to the DNA, this TF recruits a chromatin remodeling complex which displaces the nucleosome away from