Promoter-terminator fusions of E. coli ribosomal RNA operons have been constructed on multicopy plasmids in order to study regulation of RNA transcription

Abstract

Previously, we have identified a small (80bp) DNA region that when transcribed, produces anti-terminating RNA polymerase transcripts. We presume this means that the RNA polymerase elongation complex passing through this region becomes modified such that ordinarily efficient terminator sites downstream are ignored. We also found that the ribosomal RNA terminator region contains a pair of terminator sites (T1 & T2) that have a special property of being able to stop even anti-terminated transcripts. We are attempting to define the DNA sequence determinants of this special property, called "super-termination".Super-termination could occur simply as a linear additive effect of two inefficient terminators or alternatively as a more complex cooperative function. By sequencing bisulfite induced mutations in the super-terminator, functions of T1 alone and T2 alone related to termination can be defined. These are compared to mutant sites found to interfere with super-termination. In all cases terminator mutants are selected using a galactokinase detector gene and sequenced using dideoxy techniques with m13 templates.In eukaryotes the existence of extremely large heteronuclear RNA transcripts suggests transcription antitermination and super-termination mechanisms may also occur.