COPY: Prokaryotic Gene Expression

Prokaryotic Gene Expression

• Key terms, including operon, cis- and trans-acting, and merodiploid, are essential for understanding prokaryotic gene expression.

Levels of Regulation

• Gene expression in bacteria can be regulated at three levels: transcriptional, translational, and post-translational control.

E. Coli Review

• The coding strand contains an open reading frame.
• A Shine-Dalgarno box (GGAGGA) exists, which is a ribosome-binding site.

Transcription

• RNA polymerase catalyzes transcription.
• Promoters are DNA sequences near the beginning of the genes which signal RNA polymerase where to begin transcription.

Promoter Recognition

• Promoters attract RNA polymerase to initiate transcription.
• Bacterial transcription initiation signals have -35 and -10 regions upstream of the transcription start site (+1).

Regulation of Transcription

• mRNA production is determined by the promoter and surrounding sequences.
• Proteins binding to DNA can inhibit or enhance transcription.
• The Lac operon in E. coli serves as an example of gene regulation.

Operon Definition

• An operon is a cluster of genes transcribed together, including a promoter where RNA polymerase binds.

Metabolism Types

• Catabolism involves breaking down larger molecules into smaller ones to obtain energy.
• Anabolism involves utilizing smaller precursor molecules and energy from catabolism to construct larger molecules.

Lactose Utilization

• Lactose presence induces expression of genes needed for its utilization.
• Lactose added to a growth medium results in a 1000-fold increase in lac permease and β-galactosidase production.

Lac Operon

• The lac operon contains a regulatory gene (lacI), a CAP site, a promoter, an operator, and three structural genes: lacZ, lacY, and lacA.
• RNA polymerase binds to the promoter to start transcription.
• β-galactosidase, permease, and transacetylase are products of the lac operon.
• Lactose converts to glucose and galactose.
• Lactose converts to allolactose, which binds the lac repressor.
• Permease actively transports lactose into the cell.

lac Genes

• The lac genes (lacZ, lacY, lacA) are transcribed together, forming a polycistronic mRNA.

Temporal Gene Expression

• Greatest expression occurs when there is high lactose and low glucose
• Regulatory gene lacI codes for a constitutive protein that binds the operator, preventing transcription.
• An inducer, allolactose, prevents the repressor from binding to the operator, allowing transcription.

lac Repressor Protein Evidence

• Mutations in lacI result in the synthesis of β-galactosidase and permease, even without lactose.
• These mutants constitutively express these proteins, regardless of environmental conditions.

lac Repression

• In a cell without lactose, the repressor protein binds to the operator, preventing the expression of the lacZ, lacY, and lacA genes.

lac Repressor Structure

• The lac repressor has DNA-binding, inducer-binding, and multimerization domains.
• It forms an active tetramer that binds to two conserved DNA sequences and loops the DNA.
• Looping prevents RNA polymerase access to the promoter.

Lac Operon and Operators

• There are actually three operators in the lac operon.
• The repressor gene is not technically part of the operon.

Lactose-Induced Derepression

• Allolactose acts as an inducer.
• Allolactose is created from lactose by Β-galactosidase.
• LacY helps lactose enter the cell.
• Lactose is converted into allolactose by β-galactosidase (LacZ).

Lac Operon Assumptions

• Operators are leaky and can be turned ON or OFF
• The repressor binds to the operator, but not covalently.
• More active repressor leads to more binding.
• There is always a residual amount of both permease and β-gal in the cell.

lacOperon in l- Cells

• In I- cells (cells with no repressor), the lac operon is always expressed, leading to constitutive expression.

Operator Mutants

• A mutant operator (Oc) results in constitutive expression, where the lac enzymes are synthesized regardless.

Cis and Trans Action

• Cis elements regulate genes on the same DNA molecule.
• Trans elements can regulate genes on different DNA molecules.
• Proteins act in trans, noncoding DNA sites act only in cis.

Merodiploids

• Partial diploid cells, or merodiploids, are used to study the lac operon.
• They can be created by introducing plasmids into bacteria through transformation.

Analyzing lac Operon Function

• To predict lac operon function, assess the functionality of the promoter, repressor, operator, permease, and B-gal.

Another One

• The study material includes examples of genetic configurations for experimental study, referring to function in the absence or presence of lactose.

Lactose Intolerance

• Lactose intolerance affects approximately 68% of adults worldwide.
• It results from a lack of lactase, leading to bacterial fermentation in the large intestine and causing symptoms like flatulence and abdominal pain.