Parameters affecting the elongation block by RNA polymerase II at the SV40 attenuator 1 in vitro

We have previously suggested that folding of the SV40 attenuator RNA 1 into two hairpin elements leads to a block of transcription elongation. Using site-directed mutagenesis, we created three templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared among the templates, in cell-free extracts. In this report, we show that at the standard temperature (30 degrees C) the elongation block occurs at elevated KCl concentrations (0.5-1.2 M KCl) while at high temperature (65 degrees C), although the transcription elongation rate increases, the block to elongation occurs at lower KCl concentrations (less than 0.5 M KCl) as well. Since under the conditions tested the extent of the elongation block is directly dependent on the stability of the hairpin structure of the RNA, as assessed by a comparison of transcription of the wild-type and mutated templates, we suggest that elevated KCl concentrations and high temperatures are favored conditions for optimizing the processes whereby the hairpin structures are recognized by the polymerase as an attenuation signal. Moreover, the present experiments indicate that cellular elongation factors are not directly involved in modulating the extent of the elongation block at the SV40 attenuator 1 in vitro. The SV40 attenuator 1 is the only eukaryotic system known to date which has been shown to have structural characteristics so similar to rho-independent terminator in prokaryotes. We discuss the similarities between the mechanisms which lead to elongation blocks at these eukaryotic and prokaryotic sites.