RNA folding during transcription by T7 RNA polymerase analyzed using the self-cleaving transcript assay

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Polymerase Function

Background:

• RNA folding is crucial for biological function.
• T7 RNA polymerase transcribes RNA molecules.
• Hammerhead ribozymes are RNA molecules with self-cleaving activity.

Purpose of the Study:

• To investigate the length-dependent folding of RNA during transcription by T7 RNA polymerase.
• To determine the minimum RNA length required for hammerhead domain formation and self-cleavage within the transcription complex.
• To assess the impact of steric interactions within the T7 RNA polymerase ternary complex on RNA folding.

Main Methods:

• Utilizing a self-cleaving hammerhead RNA molecule.
• Employing chain-terminating ribonucleoside triphosphate analogues to arrest transcript elongation at specific positions.
• Comparing RNA sequencing ladders from cleaving templates with non-cleaving controls.

Main Results:

• Self-cleavage of hammerhead RNA within the T7 RNA polymerase ternary complex requires synthesis of 13 nucleotides beyond the cleavage site.
• RNA freed from the complex by heating can self-cleave with only 3 nucleotides beyond the cleavage site.
• Steric constraints in the T7 RNA polymerase complex prevent RNA structure formation until at least 10 bases from the polymerization site.

Conclusions:

• The T7 RNA polymerase ternary complex imposes significant steric constraints on nascent RNA.
• A minimum of 10 nucleotides must emerge from the polymerase for RNA structure formation.
• The RNA-DNA hybrid in T7 RNA polymerase ternary complexes is likely a maximum of 10 nucleotides long.