Gene Expression and Regulation Basics

• Gene expression can be regulated at many levels.
• Promoter: A region of DNA adjacent and upstream of a gene's transcriptional start site where RNA polymerase binds.
• Transcriptional regulators are DNA-binding proteins that can act as activators or repressors.
• Understanding how the Lac operon is regulated in bacteria is important
• Appreciate the complexity of eukaryotic transcriptional regulation.
• Enhancers are DNA sequences that control gene expression.
• Eukaryotic enhancers can be bound by multiple transcriptional regulators, including activators and repressors.
• General principles of transcription and its regulation are core concepts.
• Transcriptional regulation in bacteria and eukaryotes is distinct.
• The central dogma involves transcription of DNA into mRNA and translation of mRNA into protein.
• Gene expression is the process by which DNA sequence information is translated into a product, often a protein.
• The genome is constant in all somatic cells, but only a small proportion is expressed in any given cell type.
• Unused, untranscribed genes retain the potential to be expressed.
• Gene expression can be regulated at many levels, including transcriptional control, RNA processing control, RNA transport and localization control, mRNA degradation control, translation control, protein activity control
• Regulation at the RNA transcript level is relatively slow-acting and a common control point for long-term regulation.
• Protein activity control is fast-acting and readily reversible.
• In prokaryotes, transcription involves a coding strand and a template strand.
• Promoters are upstream sequences recognized by RNA polymerase to initiate transcription, but are not transcribed into mRNA
• Terminators are sequences recognized by RNA polymerase to stop transcription, near the stop site in the mRNA
• Promoters vary in strength of binding to RNA polymerase, influencing gene expression levels.
• Promoter activity is inhibited by transcriptional repressors and enhanced by transcriptional activators.
• Activators and repressors work together to allow sensitive transcriptional regulation.
• Transcriptional activators or repressors are transcription regulators or transcription factors.
• DNA-binding domains of transcription factors bind to unique DNA sequences with varying affinities.
• Different DNA binding domains allow for binding to different DNA sequences, ensuring specificity.
• An operon is a cluster of bacterial genes transcribed from a single promoter.
• The operator is a regulatory sequence that controls operon expression; it binds transcription regulator proteins.
• E. coli use glucose as an energy source, but can break down lactose if glucose is unavailable.
• The Lac operon genes enable E. coli to utilize lactose.
• The Lac operon is expressed when glucose is unavailable and lactose is present.

Lac Operon Regulation

• When glucose and lactose are both present, the Lac genes are off
• When only glucose is present, the Lac genes are off
• When neither glucose or lactose are present, the Lac genes are off
• When only lactose is present, the Lac genes are on
• The Lac repressor inhibits transcription when lactose is absent
• Allolactose, a lactose metabolite, inactivates the repressor when lactose is present and its levels reflect lactose levels
• The Lac promoter is weak, so even without the repressor, transcription isn't sufficient.
• CAP activates transcription when glucose is absent; when glucose levels are low, cAMP levels increase.
• Activators and repressors work together to regulate the Lac operon.

Eukaryotic Transcription

• Eukaryotic transcription is more complex than prokaryotic transcription.
• Eukaryotic cells have three distinct RNA polymerases: I, II, and III.
• RNA polymerase II transcribes mRNA (protein-encoding genes).
• Specific transcription activators or repressors bind to DNA sequences (enhancers) far from the promoter.
• Enhancers are regulatory DNA sequences bound by transcriptional regulators, usually activators.
• Enhancers function to allow transcription of a given gene in specific cells or developmental stages.
• Enhancers can be located far from the transcription start site.
• Mediator is a 24-subunit complex that acts as an intermediary between transcription regulators and RNA polymerase.
• DNA looping brings enhancers into proximity with the promoter and RNA polymerase
• Enhancers can be bound by multiple transcriptional regulators.
• Combined actions of multiple transcriptional regulators ensure precise gene expression regulation.
• Shh (sonic hedgehog) regulates anterior-posterior patterning of the limb and expression of Shh in the limb is controlled by a limb-specific enhancer.
• Ectopic expression is the expression of a gene in a place or time where it is not normally expressed. Ectopic expression of Shh causes extra digit formation.
• A single base-pair change can cause ectopic Shh expression in the limb, leading to digit duplication in mice.
• The Shh limb enhancer is conserved in mouse, human, and fish, but not in limbless reptiles.
• Loss of the limb enhancer for Shh is associated with the loss of limb in advanced snakes.