BIOL 438 Exam 2

Key vocabulary

Structural genes

Encode proteins with related catalytic or physical functions

Regulatory genes

Encode proteins that control the expression of structural genes of operons

Promoter

Binding sites of RNA polymerase, sites of transcription initiation

Operator

Binding sites for transcriptional regulatory proteins (repressors or activators)

• Usually near promoter, sometimes overlapping the promoter

Constitutive Genes

Genes that are expressed continuously

Regulated Genes

Genes that code for inducible enzymes needed only in certain environments

Negative Transcriptional Control

Shuts down (represses) transcription of a gene/operon in response to the presence or absence of a regulatory signal

Negative Control Examples

Lac operon or trp operon

Positive Transcription Control

Turns on (activates) transcription of a gene/operon in response to the presence or absence of a regulatory signal

Repressor Protein Conformations (2)

1. Binds to the operator and represses transcription

2. Protein does not bind to the operator and allows transcription

Lac Operon (consists of)

Consists of:

• Promoter, lacP

• Operator, lacO

• Structural genes lacZ, lacY, lacA

Lac Repressor

• Inhibitor (lacI) binds to operator (lacO)

• Bends DNA, prevents RNA polymerase from accessing promoter

• If allolactose is present, it binds repressor, RNA polymerase is then able to access promoter

Lac operon - lactose absent

Operon is repressed

Lac operon - lactose is present

Operon is induced

Trp operon

• Repressible

• Consists of 5 structural genes

• trpR (repressor) has negative transcriptional control

• The operon is transcribed at a high level only in the absence of tryptophan

Trp operon (consists of)

• Promoter, trpP

• Operator, trpO (binding site for TrpR)

• Structural genes trpEDCBA

Trp operon (trp NOT available) (first level of reg)

• TrpR does not bind to the operator

• Trp operon is transcribed (expressed)

Trp operon (trp available) (first level of reg)

• Tryptophan binds to the TrpR protein, which then binds to the operator and represses transcription of the trp protein

Difference between lac and trp operon

Lac operon is inducible, trp operon is repressible

Operons Regulated by Positive Control

• Have poor promoters

• Need transcriptional activators to assist binding of RNA polymerase to the poor promoters

• Have a special regulatory site on the DNA

• Operators in positively controlled operons are usually before the promoter

Positive Control - Transcriptional Activator Proteins

2 Conformations

1. binds to the operator and assists promoter recognition by RNA polymerase

2. does not interact with the DNA or RNA polymerase

Arabinose operon (consists of)

• AraB, AraA, AraD, and AraC

• Ara C is unable to synthesize any of the other 3 enzymes (AraBAD)

AraC - L-arabinose Present

• RNA polymerase can bind to ParaBAD

• Activates transcription of araBAD

AraC - L-arabinose Not Present

• DNA loop prevents RNA polymerase from binding to ParaBAD

• Suppress transcription of araBAD

AraI - How many parts does it consist of?

2 Parts

• araI₁

• araI₂

AraC - Presence of Arabinose

• Binds to araI₁ and araI₂

• Assists RNA polymerase to bind the promoter

AraC - Absence of Arabinose

• AraA binds to araI₁ and araO₂

• Folds the DNA into a “hairpin” structure → access of RNA polymerase to the araBAD promoter is impaired

Two Component Regulatory Systems

• Used by bacteria

• Regulate gene expression in response to environmental changes

What proteins are in two component regulatory systems?

1. Signal sensor protein

2. Signal transducer (aka response regulator) protein

What do two component regulatory systems change?

Expression of a specific set of genes

Sensor Histidine Kinase

• In cell membrane

• Has 2 domains - signal sensor domain and transmitter domain

• Detects changes in environment; when activated it phosphorylates itself

Response Regulator Protein

• Cytoplasmic

• Conducts signal from sensor kinase protein to final destination (receives P group)

• Binds DNA and regulates gene expression

Outer Membrane Porin Proteins

• EnvZ

• OmpR

EnvZ

• Sensor histidine kinase

• Autophosphorylates in high osmolarity

Porin Proteins (general def.)

Channel forming proteins that allow small molecules to pass through the outer membrane. This is regulated by the Two Component Signal Transduction System

OmpF

• Larger pore size than OmpC, allows 10 times faster diffusion than OmpC

• Dominant when E. coli in dilute environment (low osmolarity)

OmpC

• Favored in high osmolarity conditions

• Restricts passage of certain molecules, smaller channel than OmpF

OmpR

Response regulator that controls the expression of the ompF and ompC genes

Low Osmolarity (OmpF or OmpC)

Increased OmpF expression

High Osmolarity (OmpF or OmpC)

Increased OmpC expression

What is the function of the PhoR-PhoB system?

Allows bacteria to respond to low P availability by activating P starvation genes

PhoR

Sensor histidine kinase, detects P limitation

PhoB

Response regulator that controls the expression of P starvation genes

What happens to PhoR when P is sufficient?

PhoR kinase activity is inhibited, so the P starvation response is switched off

What activates PhoR

Low P availability activates its protein kinase activity

What happens after PhoR is phosphorylated?

PhoR transfers its P group to the receiver domain of PhoB

What does phosphorylation do to PhoB?

It activates PhoB, allowing it to regulate gene transcription

What is the overall result of PhoB activation?

Activation of transcription genes that encode things such as transport and hydrolysis of alternate P sources

Attenuation (general def.)

The premature ending of transcription

“Intrinsic” Transcription Terminators

A stretch of self complementary stem and loop structure followed by a run of 6 or more U’s in the mRNA

RNA polymerase FALLS OFF when encounters this, transcription is TERMINATED

What is the second level of trp operon regulation?

Attenuation, regulated by charged tRNA^Trp availability

What happens to trp operon in the presence of high charged tRNA?

Terminator (3-4 loop) forms → transcription stops

What happens to trp operon in the presence of low charged tRNA?

Antiterminator (2-3 loop) forms → transcription continues

How are riboswitches different from trp operon attenuation?

Riboswitches do NOT require translation of an mRNA leader peptide

Riboswitch

An RNA regulatory element that binds a specific molecule and changes its folding to regulate gene expression

What determines whether riboswitch controlled transcription continues or terminates?

The folding of mRNA 5’ leader sequence

mRNA “Thermometer” at HIGH temperature

mRNA unfolds → Shine-Dalgarno sequence is available for binding of ribosomes → translation can begin

mRNA “Thermometer” at LOW temperature

mRNA stem loops in the mRNA → covers Shine-Dalgarno sequence → translation is inhibited

Small RNA molecules (sRNAs aka ncRNAs)

May inhibit or enhance translation

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