Operon Vocabulary Feedback Allosteric Protein Promoter Gene ‘self regulatory biological system in which the output (or response) affects the input; can be positive or negative’ Feedback Allosteric Protein Promoter Gene Gene Expression oscillates between two different shapes depending upon what’s bonded to allosteric site location upstream of gene where RNA polymerase binds to begin transcription sequence of DNA that codes for proteins when segment of DNA is transcribed and translated into a functional protein

What is an Operon? A. System within a prokaryotic cell which is involved in controlling gene expression B. Operons usually contain clusters of genes which code for proteins (enzymes) that are all involved in one metabolic process Operons are turned ‘on’ or ‘off’ by a DNA switch (segment of DNA) called an operator If operator is open, then RNA polymerase can bind If operator is blocked, then RNA polymerase cannot bind D. Operon = promoter + operator + gene cluster

F. There are two main kinds of operon regulatory systems: E. Every prokaryotic gene cluster controlled by operon system; you need to understand function of these two operon systems: trp operon lac operon F. There are two main kinds of operon regulatory systems: •repressible operons (ex. trp operon) Normally, genes are ‘ON’ and presence of a co-repressor TURNS OFF gene expression •inducible operons (ex. lac operon) Usually genes are “OFF” but the presence of a particular substances (inducer) TURNS ON expression of one or more genes

Graphic Organizer Characteristic or Feature Repressible Operon Inducible Operon Definition Relationship to metabolism Repressor protein produced as Example Type of Gene Regulation

Repressible Operons (anabolic metabolic pathway) Utilizes repressor protein which binds to operator and prevents RNA polymerase from attaching to promoter for transcription Repressor protein created by a regulatory gene (regulator) which is independent of the operon with which it represses C. Repressor protein is allosteric ... has two distinct shapes D. In repressible operon, repressor protein produced by regulator is in inactive state E. What the heck does all this mean???

Repressible Operons (anabolic metabolic pathway) F. Take a look at the trp operon in action! (figure 18.20a, b ) trp operon picture: identify components of operon trp operon (layer 1) trp operon (layer 2) Trp Operon Animation (not from your textbook) G. Summarize NOW!! {as a flow chart?!} H. Generally, repressible operons are involved in anabolic pathways The creation of a product (co-repressor) activates the repressor to turn operon OFF (block binding of RNA polymerase at promoter)

Repressible Operons (anabolic metabolic pathway) Figure 18.3 Regulatory protein creates an inactive repressor. The operator is open and RNA polymerase can bind to the promoter and transcribe genes necessary to build tryptophan.

Repressible Operons (anabolic metabolic pathway) Figure 18.3 When tryptophan is present in the bacterium’s environment, tryptophan will bind to the allosteric repressor protein, change shape of protein and allow active repressor to bind to the operator and prevent RNA polymerase from transcribing genes that aren’t necessary. Don’t need to express genes to create tryptophan if tryptophan is present in environment.

Repressible Operons (anabolic pathway) Figure 18.3

Inducible Operons (catabolic metabolic pathway) A. Regulatory gene creates active repressor protein that binds to operator and therefore prevents transcription of gene cluster B. An inducer is required to ‘inactivate’ repressor and unblock operator... C. Inducers are usually small molecules that are involved in the catabolic pathway D. Lactose is inducer of lac operon. Lactose must be present in order to express genes and create enzymes necessary to convert lactose ---> glucose + galactose

Inducible Operons (catabolic metabolic pathway) E. Check it out: (figure 18.4) Lac Operon (with out lactose) Lac Operon (with lactose in cell) Lac Operon Animation (not from textbook) Another Lac Operon Animation F. How is lac operon ‘turned off’? Summarize how inducible operons work in your own words! G. Generally, inducible operons only transcribe catabolic enzymes when molecule involved in catabolic pathway is present in the environment & bacterial cell!

Inducible Operons (catabolic metabolic pathway: Figure 18.4) Regulatory gene creates an active repressor that immediately binds to the operator to prevent transcription of genes that code for protein enzymes that aren’t needed because the substrate is not present in the bacterium’s environment.

Inducible Operons (catabolic metabolic pathway: Figure 18.4) When allolactose is present, allolactose binds to the repressor, inactivates repressor and frees the repressor from the operator so now genes can be transcribed that code for enzymes that allow bacterium to metabolize lactose.

The lac Operon - review E. coli living in an environment with lactose: Glucose + Galactose E. coli living in an environment with lactose: Inducer (lactose) binds to repressor bound to operator Shape of repressor protein changes, and repressor disengages from operator RNA polymerase is now free to bind to DNA and transcribe lactose-catabolizing genes!

Operon Review Gene ON Gene OFF INDUCIBLE OPERON (catabolic pathway) Operator not blocked, RNA polymerase access promoter Gene ON INDUCIBLE OPERON (catabolic pathway) REPRESSIBLE OPERON (anabolic pathway) Gene OFF Active repressor bound to operator blocking RNA polymerase

How are inducible and repressible operons SIMILAR? Explain your answer... How are inducible and repressible operons DIFFERENT? BOTH are examples of negative gene regulation: Binding of repressor to operator STOPS genes from being expressed Explain your answer...

Characteristic or Feature Repressible Operon Inducible Operon Definition Relationship to metabolism Repressor protein produced as Example Type of Gene Regulation Genes normally ‘ON’, but can be turned off by co-repressor Genes normally ‘OFF, but can be turned on by inducer Anabolism – build stg nec. for survival Catabolism – Break  stg in envir. Active form Inactive form lac operon Trp operon Negative: repressor bound to operator prevents expression Negative: repressor bound to operator prevents expression

What is the selective advantage to regulating gene expression in prokaryotic cells?? Operons allow bacteria to adapt quickly to changes in their environment. These single-celled organisms must adapt quickly because they don’t have a lot of ATP to waste making unnecessary proteins The action of enzymes within the cell alter the raw materials present in the environment in which the bacteria lives Quick adaptation = survival & >> reproduction!

“Volume control” of lac Operon E.coli w/ lactose & high [glucose] Genes for lactose catabolism are ON B/c [glucose] is high, it is not as important to metabolize lactose as source for glucose Therefore genes are ON, but are QUIET Make less mRNA Do little transcription Make few catabolic enz. E.coli w/ lactose & low [glucose] Genes for lactose catabolism are ON B/c [glucose] is low, lots of lactose catabolism is required to generate glucose Therefore genes are ON and LOUD: Make lots of mRNA Do lots of transcription of genes Make many catabolic enzy.

How does a bacterium measure the concentration of glucose in the cell? How does a bacterium know to increase the ‘volume’ of transcription based on the amount of glucose in the cell?

Positive Gene Regulation of lac Operon Recall that genes for lactose catabolism are only expressed when lactose is present What if the cell is in an environment with an adequate supply of glucose and lactose? Which molecule is the first choice of bacteria to use to create ATP: lactose or glucose? GLUCOSE •How can the bacterium relate [glucose] to gene expression? Allosteric interaction between a protein and a molecule called cyclic AMP (cAMP)

ATP --------------> cAMP cyclic AMP (cAMP) adenylyl cyclase ATP --------------> cAMP cAMP accumulates when glucose is scarce The greater the [cAMP], the more cAMP binds to a protein called cAMP receptor protein (CRP). CRP is an activator of transcription!

cAMP and CRP cAMP binds to allosteric site of CRP and changes shape of CRP The new shape of CRP allows CRP to bind to a location upstream of the lac promoter CRP bends DNA and somehow makes it easier for RNA polymerase to bind to DNA and begin transcription The binding of CRP to DNA facilitates transcription and is therefore an example of positive gene regulation!

Positive Gene Regulation (figure 18.5) LACTOSE present & glucose scare so cAMP levels are high; LOTS of lactose-catabolizing enzymes transcribed (active CRP promotes binding of mRNA at promoter)

Positive Gene Regulation (figure 18.5) Lactose present AND glucose present so cAMP levels are low; few lactose-catabolizing enzymes transcribed

Relate operon to recombinant plasmid from transformation lab & biotechnology Cut arabinose gene with restriction enzyme, but promoter remains intact. Insert jellyfish gene downstream from arabinose promoter in plasmid... Arabinose (in agar medium) binds to operator to facilitate binding of RNA polymerase to promoter to transcribe gene. Now expression of jelly fish gene proceeds and bacteria colonies GLOW GREEN!! Is the arabinose operon inducible or repressible, based on this information? Arabinose operon is NEITHER inducible NOR repressible. Binding of the arabinose to the promoter (operator) FACILITATES the binding of RNA polymerase and enhances the transcription of the genes downstream. This is an example of POSITIVE GENE REGULATION: stg binding to operator TURNS ON gene expression!