LS50 2015 Gene Regulation I – transcriptional control • The problem to be solved o Not all genes are needed at same times/places/amounts o Gene expression needs to change to respond to environmental/contextual changes in the life of the cell • This is true for both single celled and multicellular organisms, prokaryotic and eukaryotic • Goal of these two lectures is to cover the major and best understood mechanisms that control gene expression Prokaryotic RNA Polymerase and promoters • RNA Pol - synthesizes all transcripts o Four subunits form the core: α, α, β, β’ o The “holoenzyme” (functional RNA Pol) is made of the core plus σ factor o σ factor recognizes and binds the promoter region • Pribnow Box: TATAAT consensus sequence o Part of the promoter sequence in bacterial promoters o About 10 bp upstream of transcription start site o Similar function to eukaryotic TATA box: needed for RNAP access The Lac Operon of E. coli • Prokaryotic genes are often grouped together into “operons,” which contain multiple genes under the same control system/sequences • The lac operon is the genetic locus that allows E. coli to metabolize lactose (if no glucose is around – glucose is the preferred source) • Contains promoters, operators (places for TFs to bind and regulate transcription), and some coding genes • Looks simple but is complex! o There are still aspects of its function being discovered after four decades of study • Arguably the first clearly understood gene regulatory system • Diauxic growth observed when glucose and lactose are provided à a “switch” in gene regulation might explain this • Multiple experimental approaches were used to understand how it works: o Genetics (Jacob & Monod): complementation testing à distinction between “structural” genes and “non-structural” genes o Molecular biology (Gilbert & Müller-Hill): purification of a repressive transcription factor à isolation of the first genetic control protein (a transcription factors that can bind DNA reversibly) Lac Operon function reveals general principles of gene regulation that can also apply to eukaryotes 1. Proteins are not synthesized when not needed 2. Cooperative binding of transcription factors 3. Redundancy and robustness: multiple operator sites, multiple regulatory inputs (e.g. lactose and glucose) 4. Allosteric regulation: conformational change in TFs 5. Switching potential: equilibrium (bound and unbound repressors), leakiness (bGal), stochasticity, environmental response (sugar source) 6. Regulatory mutations vs coding mutations Extavour Lecture Roadmaps: Gene Regulation Page 1 of 4 LS50 2015 7. Transcriptional regulation (e.g. LacI binding operator) vs post-transcriptional regulation (e.g. allolactose binding LacI) 8. Genetics (complementation testing) can help reveal molecular mechanism Major differences between prokaryotic and eukaryotic gene regulation 1. The Nucleus: Transcription and translation are physically separated in eukaryotes à some regulatory mechanisms, like attenuation, are specific to prokaryotes. 2. Activation vs Repression: Positive regulation may be more predominant in eukaryotes; negative regulation may be more predominant in prokaryotes (at least there are more well-understood examples of these). 3. Chromatin: In eukaryotes, chromatin structure must change in order for transcription to happen. The ground state of chromatin structure is not permissive to transcription. In prokaryotes, the DNA is “naked” (no histones). 4. Operons: Eukaryotic genes are usually not organized this way. Instead, each gene has its own control system/sequences. 5. Number of proteins coded per transcript: Eukaryotic genes are generally monocistronic. Prokaryotic genes are often polycistronic. 6. 5’ capping: In eukaryotes, a “cap” of 7-methyl guanosine is added to the 5’ end of transcripts. This does not happen in prokaryotes. 7. Splicing: Eukaryotic transcripts contain introns and exons; splicing occurs before translation. Prokaryotic genes lack introns and can be translated even before transcription begins. 8. Polyadenylation: the 3’ end of eukaryotic transcripts is polyadenylated. This does not happen in prokaryotes. • NB: there are approx. an order of magnitude more genes in eukaryotic genomes than in prokaryotic genomes. o Maybe this helps explain the increased reliance of eukaryotic genomes on positive regulation (the default state is for genes to be off) o Nevertheless, note that there is evidence for low levels of “nonspecific” transcription in eukaryotic genomes, including from apparently noncoding regions Eukaryotic RNA Polymerases • They have little intrinsic affinity for promoters à need transcription factors to activate transcription initiation • Pol I – rRNA synthesis • Pol II – mRNA synthesis • Pol III – tRNA and other small RNA synthesis • Pol IV (plants) – siRNA synthesis • Pol V (plants) – siRNA synthesis Pol II Promoters • The core promoter is 5’ to the transcription start site, and is where RNA Polymerase binds: o TATA Box: consensus sequence TATA § 25-30 bp 5’ to transcription start site § bound by TBP, which brings in TFIID – these are “basal” transcription factors that are necessary but not sufficient for transcription of all genes § these TFs are needed for Pol II binding to the promoter o GC Box: consensus sequence GGGCCC Extavour Lecture Roadmaps: Gene Regulation Page 2 of 4 LS50 2015 § Within a few hundred bp 5’ of transcription start site § Bound by Sp1 (basal transcription factor) o CCAAT Box: consensus sequence GCCAAT § Bound by CTF1 (basal transcription factor) § Within a few hundred bp 5’ of transcription start site • Enhancers: many different sequences, unique to specific genes o Contain multiple binding sites for different “regulatory” transcription factors that are specific to certain genes § Most regulatory transcription factors regulate the expression of many genes à pleiotropy o Can be anywhere relative to the core promoter (but are often within the 10 kb 5’ to transcription start site) o Often require looping of DNA Transcription Factor Structure • There are many different kinds of transcription factors, but a few DNA-binding domains are particularly common among them: o Helix-turn-helix (HTH) o Zinc finger: C and H residues bind Zn2+ ions o Leucine zipper: L residues at every other turn of the alpha helix Chromatin • Eukaryotic DNA is organized into chromatin • Main parameters that can feed into epigenetic gene regulation: o Histone modification § K acetylation and K/R methylation are the best studied modifications § Histone acetyl transferases (HAT) unwrap chromatin § Histone deacetylases (HDAC) condense chromatin o Histone replacement o Histone variation o Nucleosome occupancy/spacing/eviction o Chromatin packing o DNA methylation • “Epigenetics” is a word with a lot of baggage: o Originally meant any heritable non-coded change in gene function o Now almost always means anything to do with chromatin regulation Extavour Lecture Roadmaps: Gene Regulation Page 3 of 4 .