Western Kentucky University

Functional Characterization of Newly Identified Antiterminator RNAs

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Western Kentucky University

Abstract

Transcription is the central step in gene expression and is a heavily regulated process. Some bacteriophages control the expression of their genes by preventing transcription termination. Transcription terminators stop transcription while antiterminators convert RNA polymerase into a termination resistant form. RNA-mediated antitermination was first discovered in bacteriophage HK022. The transcripts of the phage polymerization utilization (put) sites directly modify RNA polymerase and promote terminator readthrough. New, put-like antiterminator RNAs were identified by searching DNA sequence databases. To determine if these sequences were functional, they were cloned into an antitermination reporter vector. Two different constructs were made using each put-like sequence: one contained transcription terminators upstream of a reporter gene and the other did not. Antitermination activity was estimated by calculating the ratio of reporter gene activity from a construct with terminators compared to a similar construct without terminators. Only one put-like sequence had significant antitermination activity. The sequence from E. aerogenes KCTC2190 promoted 8% readthrough. The sequence from S. marcescens AS13 promoted only 2.7% readthrough. The sequences from E. asburiae NCTC9394 and E. cloacae promoted less than 1% readthrough. These results have given insight into the sequence and structural requirements for antiterminator RNAs.

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Functional Characterization of Newly Identified Antiterminator RNAs

Transcription is the central step in gene expression and is a heavily regulated process. Some bacteriophages control the expression of their genes by preventing transcription termination. Transcription terminators stop transcription while antiterminators convert RNA polymerase into a termination resistant form. RNA-mediated antitermination was first discovered in bacteriophage HK022. The transcripts of the phage polymerization utilization (put) sites directly modify RNA polymerase and promote terminator readthrough. New, put-like antiterminator RNAs were identified by searching DNA sequence databases. To determine if these sequences were functional, they were cloned into an antitermination reporter vector. Two different constructs were made using each put-like sequence: one contained transcription terminators upstream of a reporter gene and the other did not. Antitermination activity was estimated by calculating the ratio of reporter gene activity from a construct with terminators compared to a similar construct without terminators. Only one put-like sequence had significant antitermination activity. The sequence from E. aerogenes KCTC2190 promoted 8% readthrough. The sequence from S. marcescens AS13 promoted only 2.7% readthrough. The sequences from E. asburiae NCTC9394 and E. cloacae promoted less than 1% readthrough. These results have given insight into the sequence and structural requirements for antiterminator RNAs.