lecture #13: regulation of gene expression (part 2)
TRANSCRIPT
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