Tandem transcription-termination sites in the late rightward operon of bacteriophage lambda

Abstract

A transcription termination site (designated as t′ _R2) is located between the rightward late t′ _R1 terminator and the S gene of phage λ. This t′ _R2 terminator is rightward and absolutely dependent on the rho factor, being about 45% effective in rho ⁺ E. coli and only 6% in rho ^- cells at 30°C. This 7.5-fold rho dependence of t′ _R2 is in contrast to that of t _R1 (4.5 fold, from about 81% to 18%) and the partial rho dependence of t′ _R1 (1.4 fold, from 96% to 67%). At the elevated temperature of 42°C, t′ _R2 becomes 1.5 times more leaky (with about 2.5-fold reduction in termination efficiency) than t _R1 or t′ _R1 (with only 1.1-fold reduction) in rho ⁺ hosts. The calculated joint efficiencies of t′ _R1 and t′ _R2 are 98% in rho ⁺ cells at 30°C. t′ _R2 is also active in vitro, but only in the presence of rho factor, whereas t′ _R1 is active both in the presence and absence of rho. However, the in vitro termination at t′ _R1 is enhanced about 1.7-fold by the rho factor. The properly oriented λ nutR site together with the N gene function bring about almost complete antitermination at t′ _R2 (96% effective), but incomplete at t′ _R1 (72%). The termination points at t′ _R2 are located around 532–534 bp to the right of the s′ _R startpoint of the p′ _R-initiated RNA on λ DNA (or 338–340 bp downstream of t′ _R1) and 66–68 bp to the left of the S gene, as determined by S1 mapping. The t′ _R2 termination points are located within a dyad symmetry region which, in the transcript, is able to form a hairpin structure consisting of 16 bp in the stem and 6 bases in the loop. It is proposed that t′ _R2 acts as a second terminator to block any readthrough transcription initiated at the late promoter p′ _R into the late genes of phage λ.