Transcriptional Regulation

Transcriptional Regulation

Getting started – Promotors, Sigma Factors, and

DNA-binding proteins

Promotors -10 and -35 consensus sequences

(before transcription, not start codon) -10 TATAAT – “TATA” or Pribnow Box -35 TTGACA – “T-T-GA-CA”

Altered sequence – weak promotor Sequence complementary to sigma

factor of RNA pol

Sigma factors vary First example of

global regulation – simultaneous, coordinated control of multiple genes and operons

Table 8.2 Brock 11th

DNA-binding Proteins Sigma factors σ70

Activators Repressors

Helix-turn-Helix Motif

Features of the Interaction Repressors often act

as dimers or tetramers

Each monomer has recognition domain and stabilization domain

Recognition sequence often involves inverted repeats

figure 8.8 Brock 11th

Eukaryotes feature Zinc-fingers and Leucine zippers

(figure 8.10)

Transcriptional Regulation

Let’s be positive

Positive regulators - activators

Activator binds to activator site or enhancer site upstream of promotor

Facilitate RNA pol binding to promotor Actual touching RNA pol “Melting”

Activator binding to DNA may require small molecule - inducer

Examples AraC protein binds L-

arabinose, and then the L-ara promotor

Maltose and the mal operon – (figure 8.15)

cAMP + cAMP Receptor Protein (CRP) – will be considered in detail later

Enhancer sites or activator-binding sites can occur distant from the promotor

Results in bending of DNA May result in opening of promotor double helix Bent DNA may be required for RNA pol-activator

complex to form Example NRI-P activator of the ntr regulon May involve Integration Host Factor (IHF)

protein

Transcriptional Regulation

Negative control of transcription:

Repression and induction

Importance of operator region

Protein binds operator and blocks RNA pol LexA repressor protein blocks synthesis of

DNA repair enzymes like uvrABC When DNA is damaged, RecA protein

becomes a protease that specifically degrades LexA protein

Repressor frequently interacts with small molecule (effector) Presence of small

molecule prevents transcription

Frequently involved in control of amino acid synthesis (anabolic) genes

Prevents costly synthesis of unnecessary proteins

Repression involves corepressor molecule binding to aporepressor protein

arginine (corepressor) binds (apo-)repressor that binds operator (fig. 8.13)

One level of control of tryptophan biosynthesis

TrpR protein – 11 kD, acts as a dimer, 50 copies per cell

Binds operator when tryptophan is present Autogenous regulation – also will block it’s own

synthesis

Small molecules (inducers) can bind repressor protein and prevent binding to operator

Enzymes will be synthesized only when inducer is present

Typically involves catabolic enzymes

Utilization of particular sugars

lac Operon – Simple Version

Inducer binds repressor protein and reduces affinity for operator

Actual inducer is allolactose (an isomer of lactose)

Artificial inducer is isopropyl-β-D-thiogalactoside

lac operon only transcribed if lactose is available

Transcriptional Regulation

Reduction of transcription after initiation:

Attenuation of the trp operon

Key Features of Attenuation Leader region (trpL)

occurs between promotor and first gene (trpE)

Leader region peptide requires 2 charged trp-tRNA

Inverted repeats lead to stem loop structures (including a terminator)

A second ribosome is needed (this is the secret nobody talks about)

trp mRNA Synthesis at Low [tryptophan] – 10% of full expression

RNA pol slides along DNA, making transcript

Ribosome starts translating message

Ribosome sails through region 1 containing tryptophan codons

Ribosome reaches stop codon and falls off

trp mRNA Synthesis at Low [tryptophan] – 10% of full expression

Consequences Leader peptide is

completed Region 1 is free to

pair with region 2 Region 3 is free to

pair with region 4 3:4 Stem loop is a

termination stem loop and RNA pol falls off – no mRNA!

Let’s take a closer look

Let’s take an even closer look

Shine-Delgarno

trp mRNA Synthesis at Very Low [tryptophan] – full expression

RNA pol slides along DNA, making transcript

Ribosome starts translating message

Ribosome stalls at tryptophan codon

trp mRNA Synthesis at Very Low [tryptophan] –full expression

Consequences Leader peptide is

not completed Region 1 can’t

pair with region 2 Region 2 is free to

pair with region 3 3:4 termination

stem loop does not form and RNA pol continues to trpE

Let’s take a closer look - again

Ribosome stalls here

Let’s take an even closer look

Shine-Delgarno

Shine-Delgarno

Attenuation is a widespread control mechanism for amino acid synthesis

Threonine Phenylalanine Histidine

7 straight His! No operator

needed – all attenuation control