Intragenic suppression of trans-dominant lethal substitutions in the conserved GEME motif of the beta subunit of RNA polymerase: evidence for functional cooperativity within the C-terminus.
BACKGROUND
The ubiquitous multimeric RNA polymerases contain two large, conserved subunits, of which the beta subunit has been implicated in all three stages of transcription. We have previously described a genetic system involving random, PCR-mediated mutagenesis of the region of rpoB encoding the C-terminal 116 amino acids of the beta subunit of Escherichia coli RNA polymerase and the characterization of dominant-negative mutations. This study identified the invariant motif GEME (residues 1271-->1274; Cromie et al. 1999). Starting with three of these GEME-motif lethal mutations (G1271E, G1271V, M1273V), we have selected for intragenic suppressors, located within the same 3'-region, that prevent expression of the trans-dominant phenotype.


RESULTS
We isolated a total of 24 missense mutants and a further 14 frameshift alleles (the latter generating a nested set of C-terminal deletions of the beta subunit) and studied the effect of the missense suppressors in vivo and in vitro. The majority of the second-site substitutions pinpoint highly conserved residues and were allele-specific. In contrast, one particular missense substitution (S1332P) acted on all three primary site mutations whilst not appreciably affecting assembly proficiency, suggesting motif-specific suppression. Two missense substitutions were found to perturb assembly of the beta subunit (M1232T and L1233P) and define a small conserved region (1228-->1233) adjacent to one of the active-site residues identified by affinity-labelling, H1237. The majority of primary mutations were located in three main clusters within the 116 amino acid region.


CONCLUSIONS
The importance and functional co-operativity of the three main clusters pinpointed is supported by the present isolation of suppressors of three different GEME primary mutations in the same three regions (whereas the suppressors of G1271V and M1273V are located in all three clusters, those for G1271E are all C-terminal of this residue). Moreover, the location of the suppressors suggests that the GEME and HLVDDK regions are present as alpha-helices in holoenzyme, and that functional co-operativity is through one particular face of each helix.
Malik
T
T
Institute of Genetics, Queen's Medical Centre, Clifton Boulevard, Nottingham NG7 2UH, UK.
Ahmad
K
K
Buyukuslu
N
N
Cromie
K
K
Glass
R E
RE
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005799:Genes, Dominant; D005804:Genes, Lethal; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D016296:Mutagenesis
D012321:DNA-Directed RNA Polymerases