Regulation of transcription initiation by Gfh factors from Deinococcus radiodurans

Transcription factors of the Gre family bind within the secondary channel of bacterial RNA polymerase (RNAP) directly modulating its catalytic activities. Universally conserved Gre factors activate RNA cleavage by RNAP, by chelating catalytic metal ions in the RNAP active site, and facilitate both promoter escape and transcription elongation. Gfh factors are Deinococcus/Thermus-specific homologues of Gre factors whose transcription functions remain poorly understood. Recently, we found that Gfh1 and Gfh2 proteins from Deinococcus radiodurans dramatically stimulate RNAP pausing during transcription elongation in the presence of Mn²⁺, but not Mg²⁺, ions. In contrast, we show that Gfh1 and Gfh2 moderately inhibit transcription initiation in the presence of either Mg²⁺ or Mn²⁺ ions. By using a molecular beacon assay, we demonstrate that Gfh1 and Gfh2 do not significantly change promoter complex stability or the rate of promoter escape by D. radiodurans RNAP. At the same time, Gfh factors significantly increase the apparent K_M value for the 5'-initiating nucleotide, without having major effects on the affinity of metal ions for the RNAP active site. Similar inhibitory effects of Gfh factors are observed for transcription initiation on promoters recognized by the principal and an alternative σ factor. In summary, our data suggest that D. radiodurans Gfh factors impair the binding of initiating substrates independently of the metal ions bound in the RNAP active site, but have only mild overall effects on transcription initiation. Thus the mechanisms of modulation of RNAP activity by these factors are different for various steps of transcription.