Characterization of an Unusual Rho Factor from the High G + C Gram-positive Bacterium Micrococcus luteus

Abstract

A transcription termination factor (Rho) was purified from the Gram-positive bacterium Micrococcus luteus, and the complete gene sequence was determined. The M. luteus Rho polypeptide has 690 residues, which is 271 residues more than its homolog from Escherichia coli. Most of the additional residues compose a highly charged, hydrophilic segment that is inserted in a nonconserved region between two conserved regions of the RNA-binding domain of the known Rho homolog proteins. This segment extends from residues 49 to 311 and includes a stretch of 238 residues that contain no hydrophobic side chains. Biochemical studies indicate that the M. luteus protein is very similar to E. coli Rho in terms of its RNA-dependent NTPase activity and its sensitivity to the Rho-specific inhibitor bicyclomycin. However, the M. luteus protein has a less stringent RNA cofactor specificity. It also acts to terminate RNA transcription with E. coli RNA polymerase on the cro DNA template, but at much earlier termination stop points than those recognized by E. coli Rho. Thus, the M. luteus protein functions as a true Rho factor, but with a different specificity than that of E. coli Rho. We propose that this altered specificity is consistent with its need to function on transcripts that have a high content of G ؉ C residues.The orderly expression of the genetic information in DNA segments into RNA molecules depends on the function of transcription terminators. In Escherichia coli, one mechanism of transcription termination is mediated in part by an essential protein factor called Rho (1). Rho factor from E. coli has been studied since its discovery nearly 25 years ago (2). The Rho monomer is a 47-kDa protein. However, Rho factor functions as a homohexamer (3) that can bind to a nascent transcript and mediate its release by actions on the transcription complex that are coupled to the hydrolysis of NTPs (1).A recent phylogenetic study by Opperman and Richardson (4) comparing rho genes isolated from organisms from several of the major branches of bacteria suggests that Rho is ubiquitous throughout the bacterial domain. An unexpected result was discovered during the analysis of the rho gene from Micrococcus luteus, a Gram-positive soil bacterium that has an unusually high G ϩ C DNA content (74%) (5). The M. luteus rho homolog was found to have an open reading frame encoding a protein that was homologous to E. coli Rho through a very long portion. However, the homology did not extend all the way through the RNA-binding domain toward the amino terminus of the protein. Because the region of homology starts in a segment that has an in-frame GTG codon preceded by a sequence that is a good match to a Shine-Dalgarno sequence, Opperman and Richardson proposed that translation began at that GTG codon to yield a 41,733-Da protein of 382 amino acids that is 52% identical (71% similar) to E. coli Rho. If this proposal were correct, the M. luteus Rho protein would be unusual in comparison with the other Rho homologs as it would lack a conserved part of its ...

Identification of an RNA-binding Site in the ATP Binding Domain of Escherichia coli Rho by H2O2/Fe-EDTA Cleavage Protection Studies

“…ATPase Assay-The ATPase activities of Rho induced by the binding of various nucleic acid ligands were determined as described by Nowatzke and Richardson (31). 80 ng of Rho was incubated with 1 mM ATP at 37°C for 5 min in 100 l of the footprinting buffer (40 mM HepesNaOH (pH 7.9), 150 mM potassium acetate, 2 mM magnesium acetate) and RNA as indicated for each reaction.…”

Structural Organization of Transcription Termination Factor Rho

“…This branch includes the Streptomycetes, Mycobacteria, and Micrococcus luteus. The rho homolog genes from three organisms in this group have been isolated and sequenced (27). They all encode larger polypeptides than do the rho genes isolated from other organisms (ϳ690 versus 420 residues) with most of the differences resulting from an insert of about 260 residues in the RNA-binding domain.…”

“…One possible adaptation that has been made in these organisms is to have a Rho factor that can initiate its action on a more highly structured RNA. Indeed, M. luteus Rho was found to terminate transcription of cro DNA with E. coli RNA polymerase at sites that are not accessible to E. coli Rho due to the structure of the transcript (27).…”

“…The M. luteus insert starts with an Ala-rich segment followed by a segment that has a large proportion of Arg, Asp, Gly, and Asn residues (27). It also has a very unusual 238-residue segment that lacks any hydrophobic residues and is thus unlikely to have ordered secondary or tertiary structures.…”

A high-affinity interaction between NusA and the rrn nut site in Mycobacterium tuberculosis

“…The mycobacterial Rho protein has not been characterized, and hence no Rho-dependent terminators have been identified in mycobacteria. However, the Micrococcus luteus Rho protein, which is highly homologous to the mycobacterial Rho proteins, recognizes the Rho-dependent terminator, tiZ1, located Ϸ200 bp into the E. coli lacZ gene (30). Assuming, therefore, that the M. smegmatis Rho protein would recognize at least one of the Rho-dependent lacZ terminators, we designed a lacZ-based construct for measuring antitermination in mycobacteria.…”