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 this gene 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 the right 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 galactose and 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 These 3 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 comes off 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 work together 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 normal lacI one mutant lacI normal lacP s normal lacO s one normal lacZ and one mutant The lacI gene is trans dominant it can bind to both operators and repress transcription in the absence of lactose When lactose is present it inactives the repressor and functional B G is produced from the good lacZ