Prokaryotic Gene Regulation

• Protein concentration is determined by regulation at multiple points
• Transcription control is a major regulatory strategy in gene expression

Learning Objectives

• Understand protein concentration is determined by multiple points of regulation
• Control of transcription is a major regulatory strategy
• Know the roles of specificity factors, repressors, and activators in prokaryotic gene regulation
• Understand the concept of operons and the roles of coding, promoter, and control regions
• Know the structure of the lac operon and the role of the cyclic AMP receptor protein in catabolic repression
• Know the structure of the trp operon, including the attenuator region
• Contrast differences in regulation with the lac operon

Protein Concentration Regulation

• Synthesis of primary transcript (transcription) increases transcript and protein levels
• Posttranscriptional modification of mRNA affects protein levels (alternative splicing)
• Messenger RNA degradation (short- or long-lived mRNAs) influences protein concentration
• Protein synthesis (translation) increases protein production
• Posttranslational processing of proteins (e.g., phosphorylation) influences protein levels
• Protein targeting and transport determines protein localization
• Protein degradation reduces protein concentration

Transcription as a Regulatory Strategy

• Transcription control is a major regulatory strategy for controlling protein synthesis
• Gene expression allows bacteria/single-cell organisms to respond and survive in their environment
• Gene expression can be constitutive (always expressed) or inducible (expression varies)
• Expression rate in inducible genes is controlled by the promoter sequence
• Regulatory proteins modulate transcription

Initiation of Transcription Regulation

• Specificity factors alter RNA polymerase specificity for promoters, e.g., sigma subunit replacement
• Repressors block RNA polymerase access to the promoter by binding to the same region
• Repressor binding sites in bacteria are called operators
• Activators enhance RNA polymerase binding, acting as positive regulatory molecules

Gene Expression Control in Prokaryotes

• Functionally related proteins' coding sequences are clustered into operons
• Operons contain coding regions, promoter regions (RNA polymerase binding sites), and control regions
• Lac operon utilizes repressors and activators to modulate RNA polymerase binding
• Trp operon includes an additional control region known as the attenuator

Lac Operon

• The β-Galactosidase is an example of an inducible enzyme
• Sequences coding for functionally-related proteins are clustered into transcriptional units called operons
• β-galactosidase catalyzes lactose hydrolysis into galactose and glucose
• Lactose addition to a culture induces β-galactosidase production
• An inducer is present for activating the operon and the inducer is allolactose and not actually lactose
• Allolactose converts lactose via transglycosylation
• Breakdown of lactose into galactose and glucose
• Digests lactose to form galactose/glucose

Lac Operon Structure

• Jacob and Monod's operon model describes transcription regulation
• Contains a regulatory gene (I), operator site (o), and structural genes (Z, Y, A: beta-galactosidase, galactoside permease, thiogalactoside transacetylase)
• Regulatory gene codes for a repressor that binds to the operator site, preventing RNA polymerase binding to the promoter
• Early models proposed that an inducer binds the repressor, blocking its operator recognition and enabling RNA polymerase transcription
• CAP (catabolic activating protein, or CRP) is required for catabolic operon activation, increases binding affinity of RNA polymerase by 50-fold

CRP's Role

• CRP is also known as a Cyclic AMP Receptor Protein and is responsible for catabolic activation
• Transcription in catabolic operons is repressed when glucose is present
• The synthesis of enzymes to metabolize other energy sources would be metabolically wasteful, so catabolism is repressed when there is plentiful glucose
• Only CRP complexed with cAMP will bind to DNA
• When glucose is high, cAMP levels will be low
• Low levels of CRP-cAMP leads to no catabolic activation of energy sources
• When glucose is low, cAMP levels will increase
• More CRP-cAMP, leads to catabolic activation of energy sources

Transcriptional Control by the Lac operon

• Note that Transcription only occurs if both conditions are met, first lactose must be present, and secondly glucose must be absent
• When lactose is absent and glucose is either present or absent, there is no transcription
• This happens because, in the absence of lactose, the repressor is active and the repressor binds to the operator
• CRP-cAMP has no role because the RNA polymerase cannot proceed across the repressor to transcribe the structural genes
• Lactose present and glucose present, there is no transcription
• Repressor binds to allolactose which changes conformation to stop it from binding the operator
• With glucose present there will be low levels of CRP-cAMP
• Without CRP-CAMP, the lac promoter is too weak to transcribe as the structure is unfavorable.
• Lactose present and glucose absent, there is transcription
• Repressor binds to allolactose.High levels of CRP-CAMP binds to the lac promoter so the conditions are correct
• Because the repressor is not bound at the site of the operator, and the favorable conditions due to glucose being absent, RNA polymerase will travel through the operator to transcribe the structural genes

Tryptophan Operon Key points

• Polycistronic mRNA codes for five enzymes (A-E) required for tryptophan synthesis
• Promoter (P) and operator (O)
• if tryptophan is absent the system will be in the ON position and therefore there will be transcription
• In the absence of tryptophan, the trp repressor has decreased affinity for the operator
• transcription can then occur
• In the presence of tryptophan, Trp binds to the trp repressor, and the affinity of the repressor for the operator increases
• The binding of this complex (tryptophan+repressor) prevents RNA polymerase binding to the promoter, blocking transcription

Trp Operon Control

• There is a 162 bp leader sequence upstream of the trp E coding region
• An attenuator region is present
• High tryptophan levels: the attenuator prevents the passage of RNA polymerase, decreasing transcription
• Low tryptophan levels: the attenuator allows more RNA polymerase to pass, increasing transcription

Trp Attenuator Mechanism

• This mechanism is dependent on the coupling of transcription and translation in bacteria
• After RNA polymerase transcribes the 5' leader, a ribosome binds and translates when encountering an AUG codon, producing a 14-residue polypeptide
• High tryptophan: the ribosome translates sequence 1 and blocks sequence 2 before sequence 3 is transcribed.
• Leads to transcription attenuation with a stem-loop structure, or a terminator attenuator structure

Trp Attenuator Mechanism

• The initiation of transcription is controlled by a repressor
• With TRP present the conditions are right to bind repressor that binds to the _trp O_perator gene
• Because it has bound to this there is no transcription
• When Trp is low repressor does not the trp does not bind the _trp O_perator gene.
• This binding of the repressor to the _trp O_perator gene leads to transcription occuring
• The ribosome pauses at Trp codons if there is low tryptopan present in sequence 1
• The paired structure formation between sequences 2 and 3 stops attenuation
• Because there is a pairing, sequences 2 and 3, it stops structure forming number 4
• Because there is a 2:3 structure, unlike the 3:4 structure, transcription can continuously occur if tryptophan is a required metabolite.