Effects of the Antiterminator BoxA on Transcription Elongation Kinetics and ppGpp Inhibition of Transcription Elongation in Escherichia coli

Ulla Vogel,Kaj Frank Jensen

doi:10.1074/jbc.270.31.18335

Ulla Vogel, Kaj Frank Jensen

Open Access

https://doi.org/10.1074/jbc.270.31.18335

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Abstract

It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of approximately 40 nucleotides (nt)/s during steady state growth on minimal medium and that the rate of mRNA chain elongation is inhibited by ppGpp in vivo. On the other hand, it was found that a truncated ribosomal RNA chain was elongated at a rate of approximately 80 nt/s, independent of growth condition (Vogel, U., and Jensen, K. F. (1994) J. Biol. Chem. 269, 16236-16241). We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons. To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes. We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s to 69 nt/s during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response. On the other hand, deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool. These results are consistent with the hypothesis that the antiterminator boxA is a primary determinant of the rate of transcription elongation rate.

Highlights

Effects of the Antiterminator BoxA on Transcription Elongation Kinetics and ppGpp Inhibition of Transcription Elongation in Escherichia coli*
We found that insertion of boxA in front of the coding region of lcreZ increased the transcription elongation rate from 42 ntis to 69 ntis during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response
To distinguish between these nonexcluding alternatives, we have studied the effects on transcription elongation of removing the antiterminator boxes from a ribosomal operon and of inserting an antiterminator boxA sequence in the early transcribed regions of infB and lacZ

Summary

Introduction

Effects of the Antiterminator BoxA on Transcription Elongation Kinetics and ppGpp Inhibition of Transcription Elongation in Escherichia coli*. We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the IcreZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes. Deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool. When Escherichia coli is exposed to amino acid starvation or other nutritional down-shifts, it undergoes the so-called stringent response, meaning that the synthesis of stable RNA (i.e. tRNA and rRNA) stops abruptly, while bulk mRNA synthesis continues (Cashel and Rudd, 1987), at reduced rate (Svitil et al, 1993; Sorensen et al, 1994). The mechanism by which accumulation of ppGpp leads to down-regulation of tRNA and rRNA

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