Lecture 7 05-23-12 Chapter 16

Gene Expression Regulation in Prokaryotes

Central Dogma of Genetics

This is the simplified model

Two important processes: Transcription and translation

Levels of Gene Expression Regulation

Only in DNA level eukaryotic Transcriptional cells level Transcriptional activators mRNA processing RNA stability Translational level Rate of translation Post-translational Protein modification

Regulatory Genes and Regulatory Elements

Structural genes: encode proteins play a structural roles in the cell Regulatory genes: genes whose products, either RNA or protein, interact with DNA sequences of other structural genes and affect their transcription or translation. DNA binding proteins - regulatory proteins Regulatory elements: DNA sequences, where the proteins produced by regulatory genes recognize and binds. Regulation of gene expression Stimulate gene expression: positive control Inhibit gene expression: negative control

DNA Binding Proteins

DNA Binding Proteins: regulatory proteins that bind to DNA sequences and affect gene expression at transcriptional level.
Discrete functional parts Domains Domains consist of 60-90aa and are responsible for binding to DNA Motifs: simple characteristic structure within binding domain, that can fit to the major groove of DNA Distinct types based on characteristic motifs

Prokaryotic Gene Structure

Bacterial genes with related functions are often clustered together with one Single promoter and transcribed together into a single mRNA Operon: a group of bacterial functional genes that are transcribed together plus the controlling sequences. Operon organization Regulator Gene encodes regulator protein (with its own promoter) controlling the transcription of other genes Promoter Initiate the structural gene expression Operator DNA sequence binds to regulator protein to regulate transcription between the promoter and the structural genes Structural genes: encodes functional proteins

Operon Structure

The operator is a DNA sequence at the border between the promoter and the structural genes

Negative and Positive Control

Two types of transcriptional control
Negative
Regulator protein repressor Binds to DNA inhibiting transcription Negative Inducible Negative Repressible Regulator protein activator Binds to DNA stimulating transcription Positive Inducible Positive Repressible

Positive

Several variations of the above

Inducible = transcription is normally off Repressible = transcription is normally on

Negative Inducible Operon

Normally, the regulator protein is bound to the operator and prevents transcription Inducer: a small molecule, which can bind to the repressor Repressor binds operator = no transcription

Negative Inducible Operon

An INDUCER must be present to bind to regulatory protein (repressor) and prevent its binding to the operator for transcription to occur Repressor cant bind = transcription

Negative Repressible Operons

Normally transcription is on, regulator protein (repressor) is inactive and not bind to the operator
no repressor = no binding on operator = transcription

Negative Repressible Operons

Co-repressor binds to the repressor allowing it bind to the operator. Transcription stops
Product (co-repressor) present = binding = no transcription

Negative Repressible Operons

REMEMBER
Negative control regulator protein = repressor, represses transcription. Inducible: normally no transcription and can be turned on Repressible: transcript normally is on and can be turned off

Positive Inducible Operons

Transcription normally is off. The regulator protein can be activated to bind to DNA (site other than operator) and activates transcription

Positive Repressible Operons

Normally, the regulator protein binds to DNA and activates transcription. Product inactivate the regulator protein and transcription turn off.

lac Operon of E. coli

Lactose: a type of sugar, an energy source for bacteria, E. coli Lactose operon: controls metabolism of the lactose in cell - lacI: regulator gene with it own promoter repressor - lacP: common promoter for 3 structural genes - lacO: operator where the repressor binds - 3 adjacent structural genes: lacZ encodes -galactosidase, which converts lactose into glucose and galactose for consumption or converts lactose in to allolactose, which regulates lactose metabolism. lacY encodes Permease allowing lactose to enter the cell. lacA encodes Transacetylase with unknown function

lac Operon of E. coli

When lactose or allolactose is present in the medium, where the bacteria grows, all 3 genes are expressed to metabolize lactose. inducible Example of negative inducible operon How are the genes activity induced, and repressed when lactose is absent??

lac Operon- Negative Control

lac Operon- Negative control

lac Operon- Positive Control

lac Operon also shows positive regulation lacZ gene codes for b-galactosidase which cleaves lactose into glucose, which the cell utilizes and prefers, and galactose when glucose is present at high levels, regardless if lactose is present, no transcription of lac operon Because b-galactosidase is not needed until the cell uses up the glucose, and then when lactose is present, lac operon gets transcribed. CAP mechanism controls the repression of the lac operon in the presence of glucose.
Results in efficient energy use in cell. No need to metabolize lactose in the presence of glucose

lac Operon- Positive Control

CAP - Catabolite Activating Protein (CAP)
CAP binds to cyclic adenosine monophospate (cAMP) cAMP alters the structure of CAP cAMP-CAP complex must bind to the promoter region for RNA Polymerase to bind for transcription of the structural genes. Glucose regulate cAMP High glucose = low cAMP, cAMP-CAP complex is not formed RNA polymerase cant bind to transcribe lac operon Low glucose = high cAMP RNA polymerase binds to the promoter for transcription

lac Operon- Positive Control

lac Operon- Positive Control

Tryptophan trp Operon

trp operon: Negative repressible gene system 5 structural genes Produce enzymes that synthesize tryptophan Upstream is the trp promoter Regulator gene (trpR) Product = regulator protein repressor Low tryptophan levels Repressor cant bind to DNA operator RNA polymerase binds DNA transcription occurs - tryptophan synthesis Tryptophan present It binds to the repressor binds to DNA operator Repress RNA polymerase binding no transcription

Tryptophan trp Operon