L6 Regulation of Prokaryotic Transcription

Lecture Aim and Learning Outcomes

• Examine mechanisms regulating gene expression in prokaryotes.
• Identify key regulatory processes in prokaryotic transcription.
• Illustrate structural organization of bacterial operons.
• Explain transcription initiation regulation with examples.
• Describe mechanisms controlling prokaryotic gene transcription completion.

Importance of Transcriptional Regulation

• Transcriptional knowledge aids in creating artificial gene expression systems.
• Supports microbial synthesis of chemicals from renewable resources.
• Applicable in human and animal healthcare for diagnostics and therapeutics.
• Addresses antibiotic resistance by developing gene silencing drugs.

Transcription Overview

• Transcription involves RNA synthesis by RNA polymerase using DNA as a template.
• The unwound region during transcription is called the transcription bubble.
• Initiation involves asymmetric transcription; only one DNA strand serves as a template.
• Termination leads to RNA transcript release and RNA polymerase detachment.

RNA Polymerase and Promoter Interaction

Prokaryotic RNA polymerase is composed of multiple subunits and can function as a core enzyme when alone, or it can combine with a sigma factor to form a holoenzyme, essential for recognizing promoter regions and initiating transcription.

• RNA polymerase functions as a complex machine essential for the process of transcription, which is the first step in gene expression. Its multi-subunit structure consists of several components: subunit α plays a key role in the assembly of the enzyme, while subunits β and β' form the catalytic core responsible for the actual synthesis of RNA strands. Additionally, the σ subunit is vital for recognizing and binding to specific promoter regions on the DNA, thereby initiating the transcription process effectively.
• The promoter is a crucial regulatory region in the DNA sequence that plays a significant role in the initiation of transcription. It contains specific sequence motifs known as the -35 and -10 boxes, collectively referred to as the Pribnow box. These motifs are recognized by RNA polymerase and are essential for the accurate assembly of the transcription machinery at the start site. The precise arrangement of these elements can greatly influence the overall transcription efficiency, as they facilitate proper binding of RNA polymerase to the promoter region, allowing for effective gene expression.
• The strength of promoter interactions influences transcription initiation efficiency.
• Regulatory factors, which include transcription factors, can bind to specific DNA sequences and either inhibit or promote the activity of RNA polymerase, thus dynamically regulating the transcription process and gene expression levels.

Promoter Escape Regulation

• Promoter escape marks RNA polymerase's transition from initiation to elongation.
• Excessively strong promoter interactions can create a situation where RNA polymerase becomes stably bound, making it difficult for the enzyme to transition beyond the initiation phase effectively.
• Regulatory proteins can impact promoter escape and serve as control points for transcription.

Repression Mechanisms

• LacUV5 Promoter: Repressed by LacI protein; both LacI and RNA polymerase can bind simultaneously.
• This binding leads to abortive RNA synthesis rather than full transcription.
• Roadblocks from DNA-binding proteins can hinder RNA polymerase movement during transcription.

Catabolite Repression (CCR)

• CCR inhibits expression of secondary substrate catabolic systems by preferred carbon sources.
• Different regulatory mechanisms are employed in bacteria for CCR, influencing virulence factors.

Inducible Promoters

• Promoters regulated by substrates, e.g., tryptophan-activated repressors that inhibit transcription when present.

Arabinose-Regulated Transcription

• In the presence of arabinose, AraC dimerizes, facilitating RNA polymerase recruitment.
• Absence of arabinose leads to DNA looping by AraC, blocking RNA polymerase binding and transcription.

Conclusion

• Prokaryotic DNA is circular, supercoiled in the nucleoid.
• Varied regulatory mechanisms operate at different transcription stages.
• Three key regulatory molecules: repressors (block transcription), activators (enhance transcription), and inducers (trigger transcription).