Comparison of three 3′ non-coding regions in Schizosaccharomyces pombe expression vectors: efficiencies of transcription termination and mRNA 3′-end formation

Abstract

  Analysis of an established Schizosaccharomyces pombe episomal shuttle vector suggested that inefficient transcription termination was deleterious to plasmid function. We undertook a study to determine if transcription in the presence and absence of 3′-processing within a vector could affect the ability of the plasmid to transform, transcribe and translate the RNA produced. This report provides an analysis of the effects that three S. pombe 3′ non-coding regions have on the transformation and expression efficiencies of a fission yeast plasmid vector. The 3′ regions from adh1, act1 and ura4 were tested for their ability to terminate and process adh1 promoter-driven transcription of a lacZ reporter gene. Differences between the 3′-processing sequences were observed, with transcription termination mediated by the ura4 3′ region being more efficient than termination by the 3′ regions of adh1 and act1. We show that plasmids containing inefficient transcription termination signals result in readthrough transcription and reduced transformation efficiencies. In addition, the readthrough transcripts containing 3′ non-coding regions show impaired translation efficiencies. We describe an S. pombe vector (pURAS) with a high transformation efficiency that directs the production of highly translatable, discrete-sized transcripts.