Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• Tiny RNA transcripts are produced during RNA polymerase transcription initiation.
• The biological function of these abortive transcripts has remained largely unknown.
• Bacteriophage T7 gene expression involves specific promoters and terminators.

Purpose of the Study:

• To investigate the biological role of abortive RNA transcripts.
• To determine the effect of abortive initiation on transcription termination in bacteriophage T7.
• To elucidate the mechanism by which abortive transcripts influence gene expression.

Main Methods:

• In vitro and in vivo transcription assays using bacteriophage T7 RNA polymerase.
• Analysis of RNA-RNA interactions and hairpin formation at transcription terminators.
• Site-directed mutagenesis of terminator sequences and abortive transcripts.
• Manipulation of RNA polymerase concentration and ribonuclease activity in vivo.

Main Results:

• Abortive transcripts from promoter phi 10 exhibit antitermination activity on terminator T phi.
• G-rich abortive RNAs sequester C+U rich sequences in T phi, preventing terminator hairpin formation.
• Antitermination is sequence-specific, depending on hybridization with the 5' half of T phi RNA.
• Mutating T phi or abortive transcripts affects antitermination, confirming the mechanism.
• Increased abortive transcript levels enhance antitermination in vivo.

Conclusions:

• Abortive initiation is not merely a byproduct but actively regulates gene expression.
• Abortive transcripts act in trans to modulate transcription termination.
• This mechanism facilitates the expression of downstream genes in bacteriophage T7.
• The findings reveal a novel regulatory role for abortive RNA transcripts in gene expression.