Exam 4 Objectives Chapter 15 Prokaryotes The most important step in the choice of making a protein is the initiation of transcription by RNA Polymerase Know what regulation of gene expression means Gene expression is regulated to maximize growth rate and is controlled at the level of transcription Therefore bacterial gene expression is mainly controlled by small molecules nutrients from the environment Understand what an operon is and how they differ from typical monocistronic genes An operon is a polycistronic gene A polycistronic gene is when one mRNA encodes two or more proteins This differs from monocistronic genes because in these one mRNA codes for one protein Bacteria have both monocistronic and polycistronic mRNA s Be able to draw a diagram showing the positions of the major elemets of the lac Operon and the I gene I gene CAP site lac promoter operator Z gene Y gene A gene and understand the function of each of these in transcription of the lac operon DNA promoter 1 Start z gene stop Start y gene stop start a gene stop term mRNA z protein y protein a protein lac Z codes for B galactosidase lac Y codes for permease allows the entry of lac into the bacterial cell and lac A codes for transacetylase which removes toxic by products of lactose digestion Know and be able to appropriately use the terms inducible expression constitutive and uninducible Uninducible cannot make b galactosidase in the presence of lactose Inducible can make b galactosidase in the presence of lactose Constitutive makes b galactosidase all the time with or without lactose Be able to describe the regulation of the lac Operon by lactose and glucose and be able to predict the effect of mutations in the lac Operon the I gene or the CAP gene on expression of the lac Operon in the presence of lactose and or glucose Understand why binding of the repressor to the operator blocks transcription of the lac operon and how CAP activates transcription Be able to state the active sites on the lac repressor and the CAP protein By regulation of lactose when lactose the inducer is present it binds to the repressor protein One lactose binds to the repressor the repressor cannot bind to the operator this allows transcription to proceed Constitutive mutations can occur in the I gene or in the Operator In a mutant repressor gene when there is no lac present the operator binding site of the REPRESSOR is altered and transcription occurs When there is a mutant operator gene and no lac is present the OPERATOR site is altered but transcription still occurs The lac Operon is also regulated by glucose As lactose induces transcription glucose inhibits it Glucose acts through catabolite activation protein CAP which binds the lac promoter and helps RNA polymerase bind This process requires cAMP Without glucose cAMP increases and no repression occurs With glucose cAMP decreases and repression does occur If there was a mutation preventing CAP protein from binding cAMP it would make the lac operon uninducible Be able to define the term transcription factor Specific transcription factors bind to specific promoter and enhancer sequences and promote binding of RNA polymerase to TATA Transcription factors contain two functional sites one that binds to the specific DNA sequence in the promoter or enhancer of a gene and a second that activates gene expression Be able to compare and contrast the regulation of the lac Operon and the trp Operon The lac operon is an example of an inducible system of gene expression because it s default state is to be inactive Only when the right catalyst is added to the system lactose is the system activated and permits gene expression The trp operon is an example of a repressive system with two types of negative control The default state for the trp operon is to be active Eukaryotes Gene expression is regulated primarily over the course of development and in response to internal signals like hormones and not to the external signals from the environment like prokaryotes Eukaryotic promoters are much larger than prokaryotic promoters and contain many more sequences that must be bound by specific transcription factors for RNA polymerase to initiate transcription This allows for the increased regulation of gene expression that is necessary in complex multicellular organisms Be able to explain the difference between promoters and enhancers Promoters are sequences adjacent to transcription start sites that contain RNA polymerase binding site They are normally 1000 or more bases long and are binding sites for 15 20 different proteins Enhancers are sequences needed for high level of transcription and can be far from the promoter They contain multiple sequence elements Know what heterochromatin and euchromatin are Heterochromatin is a chromatin that is tightly condensed and transcriptionally silent It contains DNA that has lots of cytosine methylation and has little Acetylation on its histone proteins Heterochromatin is also found in all eukaryotic cells Euchromatin is less condensed and allows transcription There are less levels of cytosine methylation in euchromatin Histone deacetylation and DNA methylation are involved in the inactivation of gene expression during differentiation Actively expressed genes have acetylated histones and have little cytosine methylation euchromatin Deacetylation of histones represses transcription and in heterochromatin transcription is permanently inactivated and has acetylated histones and highly methylated DNA The condensation of chromatin for regulation of transcription involves activator or repressor proteins that bind specific DNA sequences and attract enzymes that acetylate or deacetylate histone proteins or that methylate cytosine bases Be able to explain how steroid hormones activate the expression of specific genes in specific cells Only certain cells make the hormone receptor and this receptor is a transcription factor which when it has bound the hormone will activate the transcription of genes that contain the right DNA sequences in their promoters Cell differentiation is the production of difference cells that express different transcription factors and thus make a specific set of proteins Each differentiated cell type makes a characteristic set of proteins Chapter 17 Recombinant DNA Be able to define recombinant DNA Recombinant DNA are DNA molecules from different organisms joined together in the test tube Know what restriction enzymes do what organisms have them and what a
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