15. Operons

trp operon is a classic example of operon

negatively regulated by a repressor

trp is a biosynthetic (anabolic) operon

• synthesis of tryptophan when needed

• repressor protein is aporepressor - not active in absence of corepressor

five structural genes code for enzymes responsible for synthesizing L-tryptophan transcribed from

a single promoter negatively regulated by the TrpR aporepressor

when Trp is needed, has to be synthesized, to do so:

1. promoter available to RNAP structural genes are transcribed

2. aporepressor cannot bind to operator site

OUTCOME: structural genes TRANSCRIBED

when trp is not needed anymore because there is enough of it, synthesis has to stop

1. tryptophan (corepressor) binds to aporepressor dimer to form functional repressor dimer

OUTCOME: autoregulation repressor dimer inhibits transcription of trp operon and transcription of the TrpR gene

active repressor + inducer (lactose)

inactive repressor

OUTCOME: genes transcribed (hence inducing transcription)

aporepressor + corepressor (trp)

= active repressor

OUTCOME: no genes transcribed

L-ara operon was the first example of

positive regulation in bacteria

L-ara operon summary

• utilization of 5-carbon sugar L-arabinose - catabolism

• three structural genes (araA, araB, araD) transcribed from a single promoter PBAD

• two operators araO1 and araO2 - repression

• activated through cAMP-CAP as well

AraC regulator - repressor

binds at a one set of cis elements upstream of promoter

Arac regulator - activator upon binding of arabinose

binds at a different set of cis elements upstream from promoter

when no arabinose present

araC binds to araO2 and araI1, bends DNA and hides araPbad promoter from RNA polymerase

OUTCOME: no synthesis

when arabinose present

arabinose binds to araC, this changes the conformation of the protein, so arabinose/araC now binds to araI1, and araI2 and not to araO2

OUTCOME: araPbad promoter is available to RNAP

ara operon also subject to activation by cAMP - CAP

too much arac protein (autoregulation)

binds to araO1 → prevents furhter transcription - RNAP binds to araPc

allosteric change in regulatory molecules as a result of small molecule binding is not the only way in which regulator proteins are controlled

1. OxyR (transcriptional activator of genes induced by H2O2) is directly activated by oxidation

2. phosphorylation of regulatory proteins is a common mechanism

multiple operators - auxiliary operators in prokaryotes

strongest - most commonly shown O1

downstream O2

upstream O3

if all three O1,2,3 are occupied - trancription suppressed 1000-fold

1. if either O2 or O3 are open - 500 fold

2. both O2 and O3 open (O1 only suppressed) - 20 fold

repressor binds as a tetramer - could bind to all four of them (prokaryotes)

DNA looping - RNAP prevented from binding to promoter

presence of auxiliary operators (O2 and O3) near the functional operator O2 increases the local concentration of the repressor, so that it can occupy the functional operator O1

if a molecule cannot be transported across the membrane

two component regulatory system

1. one protein is the SENSOR-TRANSMITTER protein

   • monitors specific changes int he environment (level of nutrients. pH, solvent concentration-osmolarity etc) kinase

2. second protein is RESPONSE regulator protein

   • either stimulates or represses regulation of specific genes

   • changes in gene expression necessary for bacterium to adapt to environmental change

sensor-transmitter protein spans across the cell membrane

snesor is an outer part which detects specific changes in environment

transmitter is an inner part which usually acts as a kinase

changes in the environment - detected by sensor causing change of conformation of sensor domain causing

activates transmitter’s kinase activity in the cell

activated kinase - autophosphorylation of the transmitter domain (transfers usually gamma phosphate from atp to itself)

1. change of conformation of transmitter domain

2. phosphate is transferred to receiver domain of response regulator → change of conformation - activation of the effector domain

3. response regulator binds to DNA regulator sequences

   • transcription factor responsive to environmetnal change

direct contact between RNAP and regulator protein could

happen even when binding sites are far apart

RNAP - NtrB/NtrC nitrogen regulation system

1. RNAP binds promoter - closed complex + omega 54 factor

2. signal for low nitrogen/glutamine

3. triggers two-component regulator system

   • NtrB = sensor kinase → phosphorylates NtrC (the activator protein)

   • phophorylation changes NtrC to its active form

4. NtrC binds to enhancers far upstream (-140 and -108) from promoter

5. DNA bends so NtrC can physically contact RNAP at the promoter

6. The ATPase activity of NtrC stimulates the polymerase to unwind the template and form open complex

RNA polymerase initiates transcription at a unique site promotoer - asymmetrical

RNAP is positioned so it can transcribe only one strand from one promoter

regulatory proteins and RNA polymerase work together to regulate transcription initiation

distribution and actual nucleotide sequence of binding sites for regulatory proteins

Binding sites

usually not perfect inverted repeats - proteins bind as dimers

two basic methods that have led to understanding of transcriptional initiation and control in bacteria

1. gel shift or electrophoretic mobility shift assay (EMSA)

2. DNase I/DMS footprinting

principle of gel shift assays

electrophoretic mobility of naked DNA fragment and DNA fragment carrying bound proteins will differ, need proteins to retain 3D structure to bind to DNA

DNase I footprinting

DNase I cleaves DNA randomly, but not where a protein is bound - that bound region is “protected” and leaves a blank space on a gel

the missing bands in the protein lane indicate the exact nucleotides protected by the protein

NOTE: DNA is run on Polyacrylamide gel finer resolution = sequencing gels

DNA foot printing

part of DNA carrying protein will be protected from modification

→ carry out modification (limited DNAse digestion)

→ DNA within the protein’s footprint will be unaffected (protected)

Domains

protein primary, secondary and tertiary structures allow amino acid side chains to form regions that “read” DNA sequence

Domains recognize hydrogen bond

acceptors (oxygen and nitrogen ions) and donors (hydrogen bound to acceptor)

proteins have to recognize base pairs while

DNA is double strandedn

usually 10-20 contacts

specific H-bond donors and acceptors on protein and DNA complement each other

DNA and protein interxn not optimum simply due to sequence recognition by regulatory proteins

• maximum protein to DNA contact when DNA is distorted

• these proteins can bind and ‘bend” DNA: toward the bound dimer or away from the bound dimer

e.coli domains

the tertiary structure of large proteins is organized in distinct regions of the protein; each domain is responsible for different function (s)

motifs

specific combinations of secondary structures, which are organized into specific 3D structure inside the domains that account for the function

Transcription factors

1. dna binding domain

2. effector binding domain

3. oligomerization domain

DNA binding domain

part of the protein responsible for binding DNA, with structural motifs that read DNA sequence (the part of DNA binding domain which actually comes in contact with DNA)

effector binding domain

region of protein can be altered by binding a small molecule or covalent modification at effector site (star) causes conformational change in TF

oligomerization domain

structure to allow specific dimerization with similar TFs

DNA binding domains of regulatory proteins share

similar structural motifs

most frequent motif in DNA binding domain of bacterial repressors is

helix-turn-helix motif, about 20 amino acids long

• 2 short alpha helices (7-9 aa long) connected with a short turn

DNA recognition helix (binds specific DNA sequence) makes most of the contact with the DNA and the other stabilizes the interaction

• recognition heli and stablizing helix (clier to N terminus) forms ~ 90 degree angle

• first recognized in prokaryotes

in helix -turn-helix motfi which helix is stabilizing and recognixing

recognizing helix is #3 and stabliizing is #2,

• the N-terminal arms of the dimer’s monomers wrap around to the other face