Microbial Genetics MICB404, Spring 2008 Lecture #23 Global Regulation.

Microbial Genetics

MICB404, Spring 2008Lecture #23

Global Regulation

• Announcements– Guest lecture in the works

Dr. Stacey Gilk will discuss work she’s done with protists. Genomic/genetic-directed therapeutic drug development.

- Study guide posted- Supplemental reading material posted

Storz and Haas (2007) A guide to small RNAs in microorganisms

• Today’s lecture– Global Regulation I

• http://www.affymetrix.com/technology/index.affx• http://www.nimblegen.com/technology/

100 masks per chip (25-mers)

Mo Bio microbialgDNA isolation

kit

gDNA

FragmentedDNA

DNaseItreatment

Terminalbiotinylation

Fragmented, Biotin-labeled

DNA

RMLchipExpression

Array

TargetHybridization

Washing andStaining

Scanning andData Analysis

Image adapted from www.affymetrix.com

Comparative genome hybridization - CGH

• Phi29 polymerase

Translational Regulation

• Differential translation- Downstream genes in a polycistronic mRNA are translated less frequently than genes closer to the 5’ end (genetic polarity)

- lacZ Y A translated in a ratio of 10:5:2

- integrons, gene shuffling may determine expression level

Translational Regulation

• Genetic polarity - why?- Genes at the beginning of an operon are available for translation first, often before distal genes are even transcribed.- Rho termination factor

Posttranslational Regulation

• Protein degradation- cI in lambda- Sigma 38,

ClpXP/RssB (P+, P-)- Sigma 32,

DnaK

Posttranslational Regulation

• Protein degradation - N-end rule model: The a.a. at the N-terminus acts as a signal for proteases

N-terminal amino acid Half-life

Met, Ser, Ala, Thr, Val, Gly >20 hours

Ile, Glu 30 min

Try, Gln 10 min

Pro 7 min

Phe, leu, Asp, Lys 3 min

Arg 2 min

Posttranslational Regulation

• Protein degradation - PEST model: Determined by regions

rich in one of four amino acids.

• Proline

• Glutamic acid

• Serine

• Threonine

Tend to be degraded in less than 2 hours

Posttranslational Regulation

• Protein structural change- Protein may be activated or deactivated by other factors

- LacI/lactose- AraC/arabinose- TrpR/tryptophan

Posttranslational Regulation

• Feedback inhibition- The end product of a pathway inhibits the

activity of the first enzyme in the pathway.- Valine sensitivity- Valine and isoleucine are synthesized by the same pathway- The first enzyme in the pathway, acetohydroxy acid synthase, is feedback

inhibited by valine.

Regulatory RNA

• sRNA (trans-acting)- mechanisms

- Basepairing with target mRNA- Antisense RNA

- translation repression/activation

- mRNA stability/degradation

Regulatory RNA

• sRNA (Trans-acting)- mechanisms

- RNA-protein complexes- signal recognition, protein

secretionSecretion recognition protein

(SRP)4.5S RNA + Ffh

Regulatory RNA

• sRNA (Trans-acting)- mechanisms

- Structural mimics- 6S RNA binds to sigma 70

RNAP holoenzyme by resembling a promotor

- doesn’t bind sigma 38 RNAP holoenzyme- stationary phase

Regulatory RNA

• sRNA (cis-acting)- riboswitchesmetabolite-binding mRNA

- mRNA conformational changesmodulate geneexpression

- aptamers

Global regulation

• Adaptation and response to changing environmental conditions

• Cellular mechanisms involving multiple genes & operons– Coordinated regulation

• Regulon – A set of operons that are all regulated by the same regulatory gene

• Stimulon – A collection of operons and regulons that respond to the same environmental conditions

• Mechanisms– Transcriptional regulators

• DNA binding proteins– Ligands: inducers, co-repressors, etc.

• Activators• Repressors

– Sigma factors• RNA polymerase subunits

– alter promoter specificity of RNAP

Global regulation

• Mechanisms– Regulatory RNAs

• Transcriptional termination

• Protein binding• Anti-sense RNAs

– base-pairing to mRNA – affect translation– affect mRNA stability

Global regulation

Global regulation

StimulusPhysicalChemicalBiological

Response (Gene expression)InductionRepression

• Mechanisms– Two-component systems

• Sensor• Messenger

• Regulate a wide variety of cellular responses, including:

– osmoregulation– chemotaxis– sporulation– antibiotic production– pathogenicity

Global regulation

• Mechanisms– Two-component systems

• Sensor– Histidine kinase

• Messenger– Response regulator

Global regulation

>50 in E. coli

10 sub-families, based on additional signal output domains that they employ

Two-component systems

• Sensor– Membrane-anchored– Senses environmental stimulus– ATP-dependent autophosphorylation of

histidine residue• phosphoryl group transferred to aspartic

acid residue in response regulator

– Activation of regulator results in change of protein function or gene expression

Two-component systems

Two-component systems

Structural motifs within different types of transcription factors.

DNA Binding Domains – Conserved Motifs

Zinc Finger Motif

Helix-turn-Helix Motif Helix-loop-Helix Motif

Leucine Zipper Motif

DNA Binding Domains

Leucine Zipper Motif Helix-turn-Helix Motif

Zinc Finger Motif

Nitrogen Assimilation

• Cellular roles of nitrogen– amino acids– nucleotides– vitamins/cofactors

• Sources of N– NH3, NO3

-, organic nitrogenous compounds• N2

• Utilized at NH3 oxidation state

Nitrogen Assimilation

glnA

The amount of N available is sensed through the level of glutamine

• PII –signal transduction protein

Ntr Regulon

Ntr Regulon

• Glutamine synthetase requirement depends on N availability– glnA regulated according to [Gln]– Operon: glnA-ntrB-ntrC

• NtrB is sensor histidine kinase - autophophorylation

• NtrC is a transcriptional regulator

• Transcribed from σ54-dependent promoter– Nitrogen-responsive sigma factor– Promoter p2

» 2 other promoters, p1 and p3

• σ54-dependent promoters– NtrC~P recruits σ54-RNAP to glnA operon

Ntr Regulon

Ntr Regulon

Ntr Regulon

•At low Glutamine synthetase conc.:•NtrB autophosphorylates•NtrB~P then transfers ~P to NtrC•NtrC binds to glnA operon UAS to activate transcription

Ntr Regulon

•Low [Gln]

•PII protein modified by UMP

•NtrB free to autophosphorylate•High [Gln]

•GlnD activated

•cleaves PII-UMP bond

•Free PII binds NtrB and inhibits

autophosphorylation •No phosphoryl transfer to NtrC response regulator

•glnA operon not induced

Ntr Regulon

3 promotersp2: NtrC~P and σ54-dependent, activated at low [Gln]

p1: σ70-dependent, active at high [Gln]: expression of glnA

p3: σ70-dependent, active at high [Gln]: expression of ntrBC

glnA operon

Ntr Regulon

• NtrB– sensor histidine kinase

PII PII-UMP

low NH3high NH3

PATP binding

Ntr Regulon

• NtrC– response

regulator– transcriptional

activator

NtrC

Ntr Regulon

~P NtrB

PII-UMP PII

Low NH3 High NH3

Low GlnA High GlnA

• Wednesday’s lecture:– Global Regulation II– Reading

• Snyder and Champness, Chapter 13