A Spring-Loaded State of NusG in Its Functional Cycle Is Suggested by X-ray Crystallography and Supported by Site-Directed Mutants

• Published in: Biochemistry (Easton) 2003-03, Vol.42 (8), p.2275-2281
• Main Authors: Knowlton, J. Randy, Bubunenko, Mikhail, Andrykovitch, Michelle, Guo, Wei, Routzahn, Karen M, Waugh, David S, Court, Donald L, Ji, Xinhua
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution - genetics; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - physiology; Crystallography, X-Ray; Dimerization; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - physiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Nun protein; NusG protein; Peptide Elongation Factors - chemistry; Peptide Elongation Factors - genetics; Peptide Elongation Factors - physiology; Phage ^l; Phage HK022; Phenylalanine - chemistry; Phenylalanine - genetics; Protein Structure, Tertiary - genetics; rRNA; Structure-Activity Relationship; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - physiology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0272508

Transcription factor NusG is present in all prokaryotes, and orthologous proteins have also been identified in yeast and humans. NusG contains a 27-residue KOW motif, found in ribosomal protein L24 where it interacts with rRNA. NusG in Escherichia coli (EcNusG) is an essential protein and functions as a regulator of Rho-dependent transcription termination, phage λ N and rRNA transcription antitermination, and phage HK022 Nun termination. Relative to EcNusG, Aquifex aeolicus NusG (AaNusG) and several other bacterial NusG proteins contain a variable insertion sequence of ∼70 residues in the central region of the molecule. Recently, crystal structures of AaNusG in space groups P21 and I222 have been reported; the authors conclude that there are no conserved dimers among the contacting molecules in the crystals [Steiner, T., Kaiser, J. T., Marinkovic, S., Huber, R., and Wahl, M. C. (2002) EMBO J. 21, 4641−4653]. We have independently determined the structures of AaNusG also in two crystal forms, P21 and C2221, and surprisingly found that AaNusG molecules form domain-swapped dimers in both crystals. Additionally, polymerization is also observed in the P21 crystal. A unique “ball-and-socket” junction dominates the intermolecular interactions within both oligomers. We believe that this interaction is a clue to the function of the molecule and propose a spring-loaded state in the functional cycle of NusG. The importance of the ball-and-socket junction for the function of NusG is supported by the functional analysis of site-directed mutants.