Sequence-specific 1H, 13C, 15N resonance assignments and secondary structure of the carboxyterminal domain of the E. coli transcription factor NusA.

Abstract

E. coli NusA protein (EcoNusA) is involved in transcription pausing, termination, and antitermination by associating with the transcription complex (Mah et al., 2000). Sequence comparison among several bacterial species shows a common domain organization of NusA proteins, comprising an amino-terminal RNApolymerase (RNAP) binding domain, a central RNA binding domain of one S1 and two KH modules, and an approximately 160 amino acid carboxy-terminal domain present in some NusA proteins. The function of the carboxyterminal domain is still not fully understood, however biological data suggest an autoinhibitory role. RNA binding could be observed either in EcoNusA mutants lacking the carboxy-terminal 79 amino acids or in full-length, 495 amino acid EcoNusA in the presence of the bacteriophage λ N gene product or the carboxy-terminal domain of RNAP subunit α (Mah et al., 2000). Profile based sequence analysis predicts a modified five helix fold of two consecutive helix-hairpin-helix (HhH) motifs linked by a connector helix (Aravind et al., 1999). In addition to participating in RNA binding, this (HhH)2 fold is known to mediate protein-protein interactions (Shao et al., 2001), suggesting a major role for the carboxy-terminal domain of NusA in transcription modulation. Whereas the structures of the amino-terminal domain and the central part of T. maritima and M. tuberculosis NusA have been elucidated by X-ray crystallography (Worbs et al., 2001; Gopal et al., 2001), threedimensional structure information for the carboxy∗To whom correspondence should be addressed. E-mail: kristian.schweimer@uni-bayreuth.de terminal 160 amino acid domain of EcoNusA is unavailable. However, to address questions about the molecular basis of the interaction with the λ N protein or the RNAP α subunit and thus the basic principles governing processes such as transcription elongation, termination, and antitermination, a high-resolution structure of EcoNusA(339-495) is highly desirable. As an initial step, we virtually completely assigned the 1H, 13C, and 15N resonances of EcoNusA(339495) with multidimensional heteronuclear NMR and determined the secondary structure by chemical shift index analysis.