Mutational analysis of segmental stabilization of transcripts from the Zymomonas mobilis gap-pgk operon

Abstract

In Zymomonas mobilis, the genes encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase are transcribed together from the gap-pgk operon. However, higher levels of the former enzyme are present in the cytoplasm because of increased stability of a 5' segment containing the gap coding region. This segment is bounded by an upstream untranslated region which can be folded into many stem-loop structures and a prominent intercistronic stem-loop. Mutations eliminating a proposed stem-loop in the untranslated region or the intercistronic stem-loop resulted in a decrease in the stability and pool size of the 5' gap segment. Site-specific mutations in the unpaired regions of both of these stems also altered the message pools. Elimination of the intercistronic stem appeared to reduce the endonucleolytic cleavage within the pgk coding region, increasing the stability and abundance of the full-length message. DNA encoding the prominent stem-loop at the 3' end of the message was shown to be a transcriptional terminator both in Z. mobilis and in Escherichia coli. This third stem-loop region (part of the transcriptional terminator) was required to stabilize the full-length gap-pgk message.