RNA polymerase. Limit cognate primer for initiation and stable ternary complex formation

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Transcription initiation is a fundamental process in gene expression.
• Understanding primer requirements for DNA-dependent RNA polymerase is crucial for controlling gene expression.
• The T7 delta D111 template provides a model system for studying transcription initiation.

Purpose of the Study:

• To investigate the role of oligoribonucleotide length and sequence in primer function for T7 promoter transcription.
• To determine the requirements for forming a stable ternary complex during transcription initiation.
• To elucidate the impact of phosphodiester bond formation on primer activity.

Main Methods:

• Synthesis of various length oligoribonucleotides corresponding to T7 promoter regions.
• Assaying oligoribonucleotide primer activity with E. coli DNA-dependent RNA polymerase.
• Formation and stability assessment of enzyme-DNA-oligonucleotide ternary complexes using salt jump challenges.
• Systematic extension of primers with cognate nucleotide triphosphates.

Main Results:

• Oligoribonucleotides flanking the T7 A1 promoter can function as transcription primers.
• A stable ternary complex forms when the primer and augmenting nucleotide(s) enable normal third phosphodiester bond formation.
• Oligoribonucleotides with a preformed third phosphodiester bond are inactive as primers.
• Primer activity is sensitive to the addition of bases in the positive registry (e.g., AUCG).
• Similar phenomena were observed for T7 A2 and A3 promoters.

Conclusions:

• Primer length and the precise formation of the initial phosphodiester bonds are critical for transcription initiation by E. coli RNA polymerase.
• The stability of the ternary complex is directly linked to the successful formation of the nascent transcript's third phosphodiester bond.
• These findings provide insights into the mechanism of transcription initiation and the requirements for primer function.