Cell-free transcription of a mouse ribosomal-protein-encoding gene: the effects of promoter mutations

Abstract

The mouse ribosomal protein-encoding gene, rpS16, was accurately transcribed in vitro with a high-efficiency nuclear extract prepared from HeLa cells. An analysis of the relative activities of rpS16 templates containing deletions or deleterious mutations of various promoter elements indicated that the in vitro transcription system can recognize all of the promoter elements that were previously identified by in vivo transfection experiments. The importance of a polypyrimidine initiator, which spans the cap site, and an element termed C, which is located in the region conventionally occupied by a TATA-box, was also assessed with an appropriate set of mutant templates. In agreement with earlier in vivo studies, our in vitro results indicated that the initiator is the primary determinant for selecting the transcription start point. Interestingly, however, the in vitro system exhibited a strong bias for features that are not present in the natural rpS16 gene. Mutant templates that contained a purine rather than a pyrimidine at or near the cap site or that had the C element replaced by a canonical TATA-box were six- to tenfold more active than the wild-type (wt) rpS16 gene in vitro system, whereas in vivo, these mutants were expressed equivalently to the wt gene. A double mutant containing both a cap site substitution and the TATA-box replacement was about 18-fold more active than the wt gene in the in vitro transcription system. These results suggest that the in vitro system may preferentially build a functional transcription complex with components that do not normally interact with the wt rpS16 gene.(ABSTRACT TRUNCATED AT 250 WORDS)