TERMINATION-ALTERING AMINO-ACID SUBSTITUTIONS IN THE BETA' SUBUNIT OFESCHERICHIA-COLI RNA-POLYMERASE IDENTIFY REGIONS INVOLVED IN RNA CHAIN ELONGATION

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 beta' subunit of Escher ichia coli RNA polymerase that alter expression of reporter genes prec eded by terminators in vivo. Termination-altering substitutions occurr ed in discrete segments of beta', designated 2, 3a, 3b, 4a, 4b, 4c, an d 5, many of which are highly conserved in eukaryotic homologs of beta '. Region 2 substitutions (residues 311-386) are tightly clustered aro und a short sequence that is similar to a portion of the DNA-binding c left in E. coli DNA polymerase I. Region 3b (residues 718-798) corresp onds to the segment of the largest subunit of RNA polymerase II in whi ch amanitin-resistance substitutions occur. Region 4a substitutions (r esidues 933-936) occur in a segment thought to contact the transcript 3' end. Region 5 substitutions (residues 1308-1356) are tightly cluste red in conserved region H near the carboxyl terminus of beta'. A repre sentative set of mutant RNA polymerases were purified and revealed une xpected variation in percent termination at six different p-independen t terminators. Based on the location and properties of these substitut ions, we suggest a hypothesis for the relationship of subunits in the transcription complex.