Lecture #13:Regulation of Gene Expression (Part 2)

                                     

Lecture #13 Reading Assignment

Physiology & Biochemistry of Prokaryotes 3rd Ed.: Ch. 10, pp. 291; Ch. 18, pp. 496-503, 554-561, 509-518

Brock Biology of Microorganisms 11th Ed: Ch.8, pp. 221-227

Brock Biology of Microorganisms 12th ed.: Ch.9, pp. 231-234, 244-248

Article(s) to be discussed Majdalani Review (Bacterial Small RNA regulators)

Small Things Considered Essay for Lecture #13

Topics:

Transcriptional control by attenuation

Two-component regulatory systems

Quorum sensing

Bacterial small RNA regulators

Transcriptional Regulation: Attenuation

A system for regulating gene expression by control of transcriptional termination

Does not involve DNA-binding proteins

Leader Peptide Sequence of the Trp Operon

Attenuation Control of the Trp Operon

Overall Trp Operon Control

TrpR

Signal Transduction and Two-component Regulatory Systems

Sensor kinase has Histidine kinase activity

Response regulator has aspartate residue that becomes phosphorylated

In some systems, the de-phosphorylated regulator may be the active DNA-binding species.

Two-component Regulation: Phosphate Uptake System

From: Tetsch and Jung. Molecular Microbiology (2009) 73(6), 982–991

phoA: alkaline phosphatase pstSCAB: phosphate-specific transporter

phoE: outermembrane porin UgpBAEC: hexose-6-P and G-3-P uptake

Quorum Sensing: Control of the lux Operon

Bobtail squid colonized with luminescent Vibrio harveyi (1011 cells/ml)

LuxR

Vibrio Autoinducer (VAI)

ArcA/ArcB and LuxR-LuxI-mediated regulation ofbioluminescence in V. fischeri

5 redundant small RNAs (qrr1…qrr5) function to destabilize luxR transcript when there’s low VAI levels

From: Septer et al. 2012. Volume 7, Issue 11, e49590

Effect of Small RNA Regulators on Gene Expression

Bacterial and plasmid small RNAs are indicated in red and

Eukaryotic sRNAs are shown in blue

Positive interactions are indicated by arrows

Negative regulation is indicated by the bars

From: Storz G., Altuvia, S., Wassarman K. 2005. An Abundance of RNA Regulators. Annu. Rev. Biochem. 74:199-217

cis acting: coded on the opposite strand of targeted mRNA

trans acting: encoded at a location distant from the target

Mechanisms:1) Base pair

with other nucleic acids

2) Bind to and modify proteins

Advantages of small RNAs over protein Regulators

1) Small RNAs require fewer resources to make than a regulatory protein. Many small RNAs are expressed when carbon and energy are limiting.

2) Small RNAs act at the post-transcriptional level allowing a fast regulatory response.

3) cis-encoded RNA regulators evolve with the target

(Cheaper)

(Faster)

(Adapting)

General Mechanisms by which RNA Regulators Modulate Transcription

Ex.: 6S RNA

Ex.: SRA

Ex.: RepE and RNAI

From: Storz G., Opdyke, JA., Wassarman KM. 2006. Regulating Bacterial Transcription with Small RNAs. Cold Spring Harbor Symposia on Quantitative Biology vol LXXI. Pp 269-273.

Regulation of Transcription by the 6S sRNA

6S sRNA sequesters the polymerase complex during nutrient limitation, requiring the expression of genes controlled by an alternate sigma factor (sigma S)

From: Pichon C., Felden, B. 2007. Proteins That Interact with Bacterial Small RNA Regulators. FEMS Microbiol. Rev. 31:614-625.

The Structure of the Small RNA Regulator DsrA

DsrA functions to stimulate the translation of the rpoS mRNA

DsrA functions to inhibit translation of hns mRNA

Stimulation of RpoS translation by DsrA

DsrA requires RNA chaperone Hfq

Role of Hfq in Small Regulatory RNA Function

From: Gottesman, S. et al. 2006. Small RNA Regulators and the Bacterial Response to Stress. Cold Spring Harbor Symposia on Quantitative Biology vol LXXI. Pp. 1-10.

Possible Outcomes:

1) Promote base pairing between the small RNA and the target mRNA

2) Change the target mRNA’s accessibility to RNases.

3) Protect RNA from RNAase E digestion. Hfq mutants are viable but are slow growers.

From: Gottesman, S. et al. 2006. Small RNA Regulators and the Bacterial Response to Stress. Cold Spring Harbor Symposia on Quantitative Biology vol LXXI. Pp. 1-10.

Global Regulation by the Small RNA-binding Protein CsrA

Liu, M. Y. et al. J. Biol. Chem. 1997;272:17502-17510

Transmission electron microscopy of negatively stained CsrA-CsrB complexes. A, native complex

(micrograph); B, EDTA-treated preparation (digital image).

CsrA (carbon storage regulator) is a 61 amino acid protein that binds RNA

CsrB is a 250 nucleotide RNA that does not code for protein

CsrA CsrB

Expression of genes affected by CsrA (E.coli)

Pathways Neg. Regulated by CsrA: Glycogen biosynthesis

Glycogen catabolism

Gluconeogenesis

Pathways Pos. Regulated by CsrA: Glycolysis

Glyoxylate shunt

Acetate metabolism

Flagella biosynthesis

CsrA regulates CCM

CsrB and CsrC RNA: Titrators of CsrA

CsrBCsrC

CsrB and CsrC levels increase 3-fold when E. coli cells enter stationary phase

The BarA/UvrY 2-component regulatory system has been shown to regulate CsrB and CsrC expression

Regulation of CsrA

Proposed Model for Post-transcriptional Regulation of Gene Expression by CsrA and CsrB/CsrC RNA

CsrA-CsrC complex

From: Romeo, T. 1998. Global Regulation by the Small RNA-binding Protein CsrA and the Non-coding RNA molecule CsrB. Mol. Microbiol. 29:1321-1330.

Encodes glycogen biosynthesis enzymes

Proposed Model for Post-transcriptional Regulation of Gene Expression by CsrA and CsrB/CsrC RNA

Benefits of this Regulatory Strategy

(1)There is a low energy investment and short synthesis time to produce an RNA regulator.

(2) A single RNA can bind 18 regulator proteins, thereby amplifying its control

(3) Post-transcriptional control results in almost immediate changes in gene expression.

CRISPR: adaptive RNA regulation

From: Bhaya et al. 2011. CRISPR-Cas Systems in Bacteria and Archaea: versatile small RNSs for adaptive immunity. Annu. Rev Genet. 45:273-297.

This system is found in bacteria and archaea. The spacers regions can be an indication of evolution.

CRISPR: self vs non-self

From: Marraffini LA and Sontheimer EJ. CRISPR Interference: RNA-directed adaptive immunity in bacteria and archaea.

Pairing with the repeat prevents cleavage of self-DNA.

CRISPR: a tool for genome editing

Genes are targeted by creating a spacer with homology to the gene of interest.

From: NEB.com