Termination-altering amino acid substitutions in the β′ subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation

Abstract

To identify regions of the largest subunit of RNA polymerase that are potentially involved in transcript elongation and termination, we have characterized amino acid substitutions in the β′ subunit of Escherichia coli RNA polymerase that alter expression of reporter genes preceded by terminators in vivo. Termination-altering substitutions occurred in discrete segments of β′, designated 2, 3a, 3b, 4a, 4b, 4c, and 5, many of which are highly conserved in eukaryotic homologs of β′. Region 2 substitutions (residues 311-386) are tightly clustered around a short sequence that is similar to a portion of the DNA-binding cleft in E. coli DNA. polymerase I. Region 3b (residues 718-798) corresponds to the segment of the largest subunit of RNA polymerase II in which amanitin-resistance substitutions occur. Region 4a substitutions (residues 933-936) occur in a segment thought to contact the transcript 3′ end. Region 5 substitutions (residues 1308-1356) are tightly clustered in conserved region H near the carboxyl terminus of β′. A representative set of mutant RNA polymerases were purified and revealed unexpected variation in percent termination at six different ρ-dependent terminators. Based on the location and properties of these substitutions, we suggest a hypothesis for the relationship of subunits in the transcription complex.