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6/28/17 BIOLOGY 207 - Dr.Locke Lecture#5 - Introduction to gene regulation and operons in Prokaryotes. Required readings and problems: Reading: Open Genetics, Chapter 12 Problems: Chapter 12 Optional Griffiths (2008) 9th Ed. Readings: - Ch. 10 pp 351-363 Problems: 9th Ed. Ch. 10 1-5, 7, 9-12 (for lect#6, too) Campbell (2008) 8th Ed. Readings: Concept 18.1 Concepts: How are prokaryotic genes expressed? 1. The basics of protein coding genes 2. Transcriptional regulation involves the binding of proteins (regulatory proteins) to the DNA. 3. In prokaryotes, operons are groups or clusters of genes that are transcribed as a polycistronic mRNA. 4. The lac operon is a classic prokaryote gene (operon) and was first described using genetics. 5. Positive regulation of the lac operon Biol207 Dr. Locke section Lecture#5 Fall'11 page 1 6/28/17 Central Dogma - Genetic Information and Genes Information flows: DNA -------> RNA ------> Protein Gene expression involves: 1) Transcription - Information is transferred from the DNA sequence into an RNA sequence - messenger RNA. 2) Translation - Information is transferred from the mRNA to protein sequence. 3) Proteins function to carry out the final actions of expression. Enzyme activity -> structure/function of cell or organism etc. Phenotype What is a gene? Definition= Biol207 Dr. Locke section Lecture#5 Fall'11 page 2 6/28/17 Basic Types of genes: 1- Transcribed – polypeptide coding - mRNA -> translated into polypeptides e.g. - typical protein coding genes 2- Transcribed – Structural - transcribed into RNA used directly e.g. - rRNA, tRNA, snRNA (others, too). 3- Non-transcribed - Regulatory elements - serve to bind proteins - involved in DNA replication, transcription, and chromosome transmission e.g. - origin of replication, centromere, telomere Gene Regulation is frequently at the transcription step: First step -> fewest resources wasted. Status: on - RNA polymerase transcribes the DNA sequence and transfers the information to mRNA -> protein off - RNA polymerase doesn't transcribe the DNA sequence. Biol207 Dr. Locke section Lecture#5 Fall'11 page 3 6/28/17 Gene Regulation involves the activity of proteins Transcription depends upon: 1) RNA polymerase and 2) other proteins that either assist (promote/enhance) or prevent (repress/silence) transcription. 3) These proteins bind to the DNA (directly) or to proteins that bind to DNA (indirectly). Basic Structure of a protein coding gene: Biol207 Dr. Locke section Lecture#5 Fall'11 page 4 6/28/17 Prokaryote Gene Regulation - the lac operon Enzymes in the same metabolic pathway are often encoded by genes that are clustered and transcribed as a single unit Called - an operon. Transcription of mRNA- polycistronic - single mRNA that encodes multiple polypeptides - AUG start-codon to UAG stop-codon (start to stop etc.) - polycistronic mRNA; cistron = gene - provides co-ordinate control of multiple gene products (polypeptides) that are needed at the same time in the same process Only some prokaryote genes are operons – not all. Diagram of lac operon Biol207 Dr. Locke section Lecture#5 Fall'11 page 5 6/28/17 Positive regulation at the lac operon - promoter sequences – CAP and polymerase - polymerase transcribes operon - CAP protein – active complex requires cAMP - binds to DNA and to polymerase to increase level of transcription Catabolite Repression – Positive control Glucose levels in the cell affect transcription of the lac operon LOW glucose levels: Adenylate cylcase (on) ATP -------> HIGH glucose levels: Adenylate cylcase (off) ATP ----| Biol207 Dr. Locke section Lecture#5 Fall'11 page 6 cAMP cAMP 6/28/17 Genetics of the lac operon Mutations Location of mutation: Result: Not in gene Positive regulation - Structural Positive regulation - Regulatory Negative regulation - Regulatory Biol207 Dr. Locke section Lecture#5 Fall'11 page 7 6/28/17 Negative Regulation First understanding of gene regulation comes from the work of Jacob and Monod in the 1950's and ‘60's -> Nobel prize in 1965. Inducers - specific substrates that induced the appearance of specific enzymes (new synthesis of the enzymes). beta-galactosidase could be induced with several types of beta-galactosides – IPTG – Fig The enzyme is not normally present in absence of an inducer. Biol207 Dr. Locke section Lecture#5 Fall'11 page 8 6/28/17 Constitutive mutants of the I locus J & M found a mutant that produced beta-galactosidase at full level all the time (even in the absence of inducer). It is called a Constitutive mutant - always "on" - expressed - unregulated by inducers. Genetically mapped close to, but distinct from, the ZYA genes These mutants defined the I locus. I I + cells are inducible to full lac ZYA levels only in presence of inducer - mutants are not inducible but instead, always express full levels of ZYA in either presence or absence of inducer See Fig Biol207 Dr. Locke section Lecture#5 Fall'11 page 9 6/28/17 Lac operon: Fig The I gene product is a repressor protein which acts by binding to the operator DNA sequence thereby preventing the RNA polymerase (which binds to the promoter-P sequence) from transcribing a polycistronic messager RNA of the cistrons Z, Y, A. Example of negative regulation - repressor protein acts to prevent transcription - transcription would take place in the absence of the repressor. Biol207 Dr. Locke section Lecture#5 Fall'11 page 10