Transcriptional Regulation in Prokaryotes

Parts 1 and 2

Constitutive Expression: some genes are expressed in _____________

all conditions

• Required for basic cellular functions (aka “housekeeping” genes)

• Ex. genes encoding RNA polymerase, ribosomal RNAs, ribosomal proteins, cytoskeletal components (like actin), some metabolic enzymes (GAPDH)

Inducible/Repressible Expression: some genes are expressed in_________

only some conditions: 

• Single-cell organisms (prokaryotes, yeast): response to environmental and nutritional signals

• Multicellular organisms (plants, animals): cell-cell communication during development, response to environment, tissue-specific expression

Structural Genes

Encode proteins that function in metabolism, biosynthesis, or structural aspects of the cell

Regulatory genes

encode regulatory RNAs or proteins that control expression of structural genes and/or their products

• Many transcriptional regulatory proteins have DNA-binding activity

Regulatory Elements

DNA sequences that are not transcribed but play a role in regulating other genes

• Usually the site of binding of regulatory proteins

Transcriptional Regulation

• Initiation of transcription: chromatin structure and DNA methylation (eukaryotes); usually most important aspect of gene regulation in bacteria

• Shift from initiation to elongation

Posttranscriptional Regulation 

Primarily Eukaryotic

• Stability (half-life) of mRNA, exist longer can produce more protein

• mRNA processing and transport to cytoplasm

• Efficiency of translation initiation

• Stability and posttranslational modification of polypeptide

• Protein activity: allosteric regulation

Operon

set of functionally related structural genes transcribed from a single promoter

Operator

cis-acting regulatory sequence where trans-acting regulatory proteins bind and affect transcription

• regulator protein binds the operator

Many bacterial genes are organized into operons

Coordinate expression of multiple genes that are all required for a particular pathway or function

Note that the regulator protein is constitutively expressed from a separate gene and binds to the operator that is within the operon

Operon

Cluster of genes that are concomitantly controlled by the same regulatory element and regulatory protein

Controlled by regulatory proteins (positively or negatively)

What is an operon composed of?

Regulatory protein (with allosteric control), operator, promoter, structural genes

Structural genes share same regulation patterns

mRNA synthesis from operon

mRNA synthesized as one molecule

contains multiple open reading frames to make different proteins

Allosteric regulation of protein structure and function

Chemicals induce structural changes of proteins

Inhibitors can bind to allosteric site and prevent the regulator protein from binding to the regulator sequence

Activators can bind to allosteric site and allow the regulator protein to bind to the regulator sequence

Lactose metabolism steps

1. Permease actively transports lactose into the cell

2. Beta-galactosidase breaks it into galactose and glucose

3. Beta-galactosidase converts lactose into the related compound allolactose (binds to lac regulator)

4. Beta-galactosidase converts allolactose into galactose and glucose

lac operon

coordinated control of lactose metabolism

lacI

repressor gene

lacP

Promoter

lacO

operator (where repressor binds)

3 structural gene/proteins within the lac operon

lacZ: beta-galactosidase

lacY: permease

lacA: transacetylase

No lactose within the lac operon

lacI binds to lacO, lacZ/Y/A not expressed

With lactose within the lac operon

lacI falls off lacO, lacZ/Y/A are expressed

lacY

encodes permease: transports lactose into the cell

lacZ

encodes beta-galactosidase: breaks down lactose

lac promoter (lacP)

binds RNA polymerase

lac operator (lacO)

binds the lac repressor protein

CAP

Catobolite activator protein contains a binding site prior to the promoter

Trans-acting elements

Regulatory proteins encoded by regulatory genes

• DNA binding proteins (ex. lacI)

• Can diffuse through the cytoplasm and act at target sites on any DNA molecule in the cell

Cis-acting elements

Regulatory DNA elements/sequences

• Specific DNA sequences (ex. lacO or lacP) that are binding sites for regulatory proteins

• Can only influence expression of adjacent genes on the same DNA molecule

Jacob and Monad used partial diploids to dissect regulation of the lac operon

they asked:

• Which lac alleles are inducible?

• Which lac mutations act in cis and which act in trans?

• Which lac mutations are dominant to wildtype?

Partial diploid

two copies of lac sequences

• ex. lacI+, lacZ- ; lacI-, lacZ+

Structural gene mutations

Affect lacZ, lacY

• Affects only lacZ and lacY

• Alter amino acid sequence of protein encoded by gene in which mutation occurs

Regulator-gene mutations

Affect lacI

• Trans element

  • Affect transcription of structural genes

Operator mutations

Affect lacO

• Cis element

• Affect transcription of structural genes

Promoter Mutations

affects lacP

• Cis elements

• Affect transcription of structural genes

lacZ^-

Effect: nonfunctional beta-galactosidase with no effect on lac mRNA expression

lacY^-

Effect: nonfunctional permease with no effect on lac mRNA expression

lacI^-

Effect: defective repressor cannot bind to lacO, trans acting that has a constitutive effect on lac mRNA expression

lacI^S

Effect: superrepressor, lacks binding site for inducer, trans acting that has a non-inducible effect on lac mRNA expression

lacO^C

Effect: fails to bind repressor protein, cis acting with a constitutive effect on lac mRNA expression

lacP^-

Effect: defective promoter, cis acting leading to no expression of the mRNA

Negative Regulation

repressor binds to regulatory sequence and blocks transcription

Positive Regulation

activator binds to regulatory sequence and enables transcription

Inducible

presence of the effector enables transcription

repressible

presence of the effector blocks transcription

Negative inducible regulation

effector inhibits binding of repressor to regulatory sequence → activates transcription

Positive inducible regulation

effector activates binding of activator to regulatory sequence → activates transcription

Negative repressible regulation

effector activates binding of repressor to regulatory sequence → inhibits transcription

Positive repressible regulation

effector inhibits binding of activator to regulator sequence → inhibits transcription

Normal Regulation of Beta-gal expression

Genotype:

• Allele 1: lacI+ lacO+ lacP+ lacZ+

• Allele 2: lacI+ lacO+ lacP+ lacZ+

Phenotype: Normal Beta-gal regulation (without lactose → no expression; with lactose → Beta-gal expression)

Mutation of Structural Gene on Both Alleles (lacZ-)

Normal lacZ sequence is required for beta-gal activity

• Genotype: lacP+ lacZ- / lacP+ lacZ-

• Phenotype: no Beta-galactosidase activity

Mutation of Structural Gene on One Allele (lacZ-)

LacZ- is recessive mutation and can only cis act

Genotype: lacP+ lacZ- / lacP+ lacZ+

Phenotype: Normal beta-gal regulation

Mutation of Promoter on Both (lacP-)

lacP is required for structural gene expression

• Genotype: lacP- lacZ+ / lacP- lacZ+

• Phenotype: no Beta-gal activity

Mutation of Promoter on One (lacP-)

lacP can only cis act to control expression of structural gene from the same allele

• Genotype: lacP- lacZ+ / lacP+ lacZ-

• Phenotype: no beta-gal activity

Mutation of Operator on One (lacO^C)

Repressor protein cannot bind to (constitutive) lacO^C DNA even in absence of lactose, and lacO^C can only cis act

• Genotype: lacO^C lacZ+ / lacO+ lacZ+

• Phenotype: beta-gal activity always detected

Mutation of Regulator Gene (lacI-)

lacI+ can trans act to regulate expression of structural gene from a different allele

• Genotype: lacI- lacZ+ / lacI+ lacZ+

• Phenotype: normal beta-gal regulation

Mutation of Regulator Gene (lacI^S) → super-repressor

lacI^S represses lacZ expression all the time, and can trans act

• Genotype: lacI^S lacZ+ / lacI+ lacZ+

• Phenotype: no beta-gal activity

Mutation of Regulator Gene (lacI^S) cannot work on lacO^C

• Genotype: lacI^S lacO^C lacZ+ / lacI+ lacO+ lacZ+

• Phenotype: beta-gal activity always detected

cAMP-CAP

Positive regulator of lac transcription and glucose controls its binding to the CAP binding site

cAMP-CAP complex binds to the CAP binding site

Positive Regulation: cAMP-CAP binds to DNA near lacP and enhances binding of RNA polymerase to lacP

• 50-fold increase in transcription of lac structural genes

• CAP activates expression of ~ 100 different genes

• DNA binding domain is helix-turn-helix

Catabolite (glucose) control of lac Operon

• CAP needs to bind with cAMP in order to bind to DNA at lac promoter, facilitating RNA polymerase binding

• Mutations in CAP gene abolish expression of lac operon

• When glucose is high, even the presence of lactose, lac operon cannot be activated

• RNA polymerase does not bind efficiently to promoters unless CAP is first bound to the DNA

Control of lac transcription by glucose (catabolite repression example)

When the glucose level is low, cAMP levels are high (cAMP-CAP increases transcription of the lac operon)

• low glucose → high adenylate cyclase → high cAMP

When the glucose level is high, cAMP levels are low (low cAMP-CAP, minimal transcription of the lac operon)

• high glucose → low adenylate cyclase → low cAMP

trp operon structural genes

trpE, trpD, trpC, trpB, trpA

Negative repressible

trp structural genes expressed only when tryptophan levels are low

• no tryptophan: trp genes are expressed (trp repressor cannot bind to trpO and trp structural genes are transcribed)

• with tryptophan: trp genes are not expressed (trp repressor binds to the trpO → expression of trp structural genes is repressed)

Two levels of control of trp gene expression

1. trp repressor (trpR) acts on trp operator (trpO)

2. Attenuation (provides extremely rapid response to need for protein synthesize)

Trptophan is the effector and is a ______________

co-repressor

• Binding of trp causes allosteric change in trpR repressor protein and allows it to bind to the operator

trpR- mutants

still show some operon-inhibitions by tryptophan

• Tryptophan operon can produce short (141 bp) and long (~6,500 bp) transcripts

• In high tryptophan, find many short transcripts (early termination of transcription)

• In low tryptophan, most transcripts are full-length

What causes very early termination of transcription in presence of tryptophan? 

How can the level of an amino acid be linked to transcription?

Requires translation and transcription to co-occur and speed of translation to affect early transcription termination: key is in the leader sequence

trp operon leader sequence

• leader sequence contains four regions of complementary sequence

• two of these, 3 and 4, can pair to form a termination stem loop

Formation of a 3-4 step loop terminates transcription before any of the structural genes are transcribed

Formation of 2-3 stem loop prevents formation of 3-4 stem loop, and transcription continues into the structural genes

How does attenuation work?

Tryptophan Present: ribosome moves quickly, reaches region 2 before region 4 is transcribed, allowing the 3-4 stem loop to form (3-4 stem loop forms, thus early termination)

Tryptophan Absent: ribsome stalls at trp codons, region 4 is transcribed before region 2 is reached by ribosome, allowing 2-3 stem loop to form (no 3-4 stem loop forms)

Why need 2 types of trp operon control mechanisms: trpR and attenuation?

• trpR mediated repression can inhibit 70 fold

• Attenuation can further reduce transcription another 8- to 10-fold 

• Together, the two processes are capable of reducing transcription of the trp operon more than 600-fold! Higher precision control

Which components can cis-act within the lac operon?

Operator (lacO), Promoter (lacP), and Structural Genes (lacZ, lacY, or lacA)

Which components can trans-act within the lac operon?

Regulatory protein (lacI)

What are the key differences for the function/effect of cis-acting elements and trans-acting elements in partial diploid cells?

Thay can’t affect the expression of structural genes from the other allele, while trans-acting elements can

Question 1 Flow Chart

Both promoter (lacP) and structural gene are normal (+) in the allele that you are working on?

Yes → next question

No → no enzyme activity (w/ or w/o lactose)

Question 2 Flow Chart

Is there a lacO^C mutation in the allele that you are working on?

Yes → have enzyme activity (w/ or w/o lactose)

No → next question

Question 3 Flow Chart

Is there a lacI^S mutation in EITHER allele 1 or allele 2?

Yes → no enzyme activity (w or w/o lactose)

No → next question

Question 4 Flow Chart

Is there a normal lacI allele (lacI+) in either allele 1 or allele 2?

Yes → no enzyme activity w/o lactose; have enzyme activity w/ lactose

No → have enzyme activity (w/ or w/o lactose)