T7 early RNAs are generated by site-specific cleavages

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Purified Escherichia coli RNA polymerase transcribes T7 DNA into long RNA molecules.
• These long RNAs initiate near the left end of T7 DNA and terminate at the early region's end.
• Early T7 RNA molecules observed in vivo are similar to those generated by specific cleavage of long transcripts.

Purpose of the Study:

• To identify and characterize the enzyme responsible for processing long T7 RNA transcripts.
• To understand the mechanism of early T7 messenger RNA (mRNA) generation.
• To investigate the role of specific initiation sites and cleavage events in T7 RNA synthesis.

Main Methods:

• Isolation and characterization of an endonuclease from uninfected E. coli.
• Analysis of RNA cleavage products generated by the endonuclease.
• Mapping of RNA initiation sites on T7 DNA.
• Investigation of the role of factors like Rho in RNA synthesis.

Main Results:

• An endonuclease (potentially RNase III) was isolated that cleaves long T7 RNAs at five specific sites.
• These cleavages generate RNA molecules identical to in vivo early T7 RNAs.
• Early RNA synthesis initiates at three strong sites (150-200 bp apart) and some minor sites.
• Cleavage by the sizing factor produces five early T7 mRNAs and three overlapping promoter region RNAs.
• Initiation starts with Adenosine (A) at two strong sites and Guanosine (G) at the third.
• Rho factor is not required for the generation of these early T7 RNAs.

Conclusions:

• A specific endonuclease acts as a sizing factor to generate functional early T7 messenger RNAs.
• The process involves specific cleavage of long transcripts initiated at defined sites.
• This mechanism ensures the production of essential T7 mRNAs for gene expression without Rho factor involvement.