BSCI222 – Lecture 6 (9/19/13)- Control of Gene Expression in Prokaryotes (Chapter 16)- There is a general trade-off between speed of regulating gene activity and cost of regulating that activity.o If you want to turn a gene off, it’s cheapest to turn it off at the beginning of the process – but then if you want to turn it on later, it’s more expensive because you have to go through the whole process.o It’s expensive to keep a whole bunch of inactive proteins in the cells that are not doing anything.- 2000 genes in E. coli, must be able to differentially regulate. Some genes, you need a set of genes all at the same time. In bacteria, many genes are organized into operons of several genes, which are all organized into a pathway, same transcript, but can be separately translated (mRNA has operator, sequence in the promoter that binds a regulatory protein/repressor protein that can block the binding of RNA polymerase to thisgene to prevent transcription).o The regulator, once translated (from the regulator region next to the promoter), is ready and able to bind to the operator.o Inducers bind to the repressor proteins and change their shape, in such a way that they can no longer bind to the operator. Inducers stop these regulators. Typically, the inducer is a precursor (a substrate) to the transcription and translation pathway(that way when the substrate is available, it all gets cranking). o There are also different repressor proteins that when they are transcribed and translated are NOT in the right shape to bind to the operator, and only become theright shape when something binds (typically, the product of the biochemical pathway/transcription and translation pathway; that way, when we have plenty of product and don’t need any more, it stops the pathway by binding to the repressor protein and shutting the whole thing down). (That last product is called a co-repressor). Negative inducible and negative repressible.o Can also have positive regulation of the gene – something that binds a little upstream of the basal promoter and helps to attract, helps to improve the binding of RNA polymerase – called activators (positive effects on transcription). Is inducible – normally not the right shape to bind, but when the appropriate substrate is available it binds -> changes shape, binds.o Also have a positive repressible – normally active, until product binds to the activator and removes it, making it inactive and reducing transcription. (PowerPoint has a 4 square slide summarizing all this)- The lac operon (first operon that all this got worked out)o Lactose is great fuel for E. coli (disaccharide in extracellular environment)o Permease gets lactose into the cell, then beta-galactosidase breaks it into galactoseand glucose. It also converts lactose into a related compound called allolactose, and then converting that into galactose and glucose. The permease is encoded by lacY, the B-G by lacZ, and then there’s lacA (function not yet understood). These3 genes are part of the operon. Way farther up, independent of the operon, is lacI, which encodes a repressor protein, which normally binds to the lacO operator. If no lactose in the environment, then this pathway is normally repressed.o If there’s a significant amount of lactose in the environment, some will get in the cell even without permease. And even though the operon is shut down, there’s always a little bit of leaky expression. The protein is not so tightly bound that there’s never any expression. And early proteins from earlier activity are still hanging around. That low level of lactose gets into the cell -> there will be some allolactose to bind to the repressor protein (lacI), change its shape so that it comesoff the operon’s operator. This starts the machine, RNA polymerase binds, start transcription, make permease and B-G and chugging away. o The lac promoter (lacP) has typical -10 and -35. The lac repressor (lacZ) covers the start site, downstream from the promoter.o The lac operator is an “interrupted palindrome”, has symmetry. Find regions of sequence that are the same on the two strands (when read in opposite directions, as you should). This kind of symmetry in the genome typically indicates a protein acting as a dimer, binding to both sides of the gene. lacI (repressor) is indeed a dimer.o Multiple operator sequences ahead of the lacO (we’ve been talking about lacO1). There’s also 2 a little downstream, and 3 slightly upstream, of lac O. 3 and 1 worktogether. One dimer binds to #1, another dimer binds to #3, and those two dimers interact together, and pulls the two regions together, causing the DNA in between to go up in a loop, which is exactly where RNA polymerase would want to bind and start transcribing. lacO3 is -82, lacO1 is +11, and lacO2 is +412. Having multiple operators makes things more stable and makes a pretty tough off switch.o TEST: will write genotype of E. coli (lacI, Promoter, Operator, B-G gene called Z, and permease gene called Y. Each will have a + (meaning wild type or normal version of that DNA sequence) or a – (meaning defective, maybe deleted.) Then, with that genotype, the question asks will there be B-G activity (operon on or not)depending on whether there is lactose (and thus allolactose) or not.  No allolactose -> all genotypes + -> lacZ gene should be off, no B-G activity. Lactose present -> allolactose -> all genotypes -> B-G activity and transcription. Mutations -> same question. O^c (means constitutive expression, always on. Can’t bind to the repressor.). Thus transcription, and B-G activity, whether or not lactose is there because the repressor can never bind, machine always chugging away. If the promoter is defective/deleted, can RNA polymerase bind? Nope! Thus no transcription, no B-G activity, whether or not lactose is there. If everything normally but deleted lacZ, the B-G gene, then transcription and translation MIGHT be happening but no B-G activity (Tricky!), and the protein won’t have the appropriate B-G function.o What if you had two copies of the operon? Partial diploids (prokaryotes have no nucleus, this is all in the cytoplasm, which is why the lacI can diffuse around): chromosomal copy of lac operon, and a second copy of some (or all) lac genes on a plasmid, and those genes might be normal or mutant. Allows examination of cis (same strand) or trans (different strand) action on genes. Example: 2 normal P’s, one