Journal of Molecular Biology
Volume 377, Issue 5, 11 April 2008, Pages 1460-1473
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Crystal Structure and RNA Binding of the Tex Protein from Pseudomonas aeruginosa

https://doi.org/10.1016/j.jmb.2008.01.096Get rights and content

Abstract

Tex is a highly conserved bacterial protein that likely functions in a variety of transcriptional processes. Here, we describe two crystal structures of the 86-kDa Tex protein from Pseudomonas aeruginosa at 2.3 and 2.5 Å resolution, respectively. These structures reveal a relatively flat and elongated protein, with several potential nucleic acid binding motifs clustered at one end, including an S1 domain near the C-terminus that displays considerable structural flexibility. Tex binds nucleic acids, with a preference for single-stranded RNA, and the Tex S1 domain is required for this binding activity. Point mutants further demonstrate that the primary nucleic acid binding site corresponds to a surface of the S1 domain. Sequence alignment and modeling indicate that the eukaryotic Spt6 transcription factor adopts a similar core structure. Structural analysis further suggests that the RNA polymerase and nucleosome interacting regions of Spt6 flank opposite sides of the Tex-like scaffold. Therefore, the Tex structure may represent a conserved scaffold that binds single-stranded RNA to regulate transcription in both eukaryotic and prokaryotic organisms.

Introduction

The Tex (toxin expression) protein was originally described in Bordetella pertussis as an essential protein involved in expression of critical toxin genes.1 Tex is a relatively large protein with a domain architecture consisting of several nucleic acid binding domains predicted from primary sequence. The presence of these domains supports the proposal that Tex is a transcription factor that functions in toxin expression and/or pathogen fitness.1, 2, 3 Tex displays a remarkably high degree of identity and similarity across a host of significant pathogens. For example, Tex from Pseudomonas aeruginosa shares 65% identity and 78% similarity (at the amino acid level) with Tex from Vibrio cholerae (the causative agent of cholera). Similar degrees of identity are seen with Tex proteins from Shigella flexneri (the causative agent of dysentery) and Yersinia pestis (the causative agent of plague).

Despite being ubiquitous and extremely well conserved, the molecular functions of Tex remain enigmatic. Insight into Tex function is derived from several bacterial studies. Aside from its role in expression of toxin gene products in B. pertussis, the tex gene from P. aeruginosa (PA5201) appears to play an important role in pathogenesis, being required for lung infection in a chronic disease model.4 In Streptococcus pneumoniae, Tex does not alter expression of the major pneumococcus toxin pneumolysin but does appear to be a transcription factor involved in pathogen fitness.3 These studies indicate that Tex may play a role in gene expression or transcript maintenance of either specific toxin or general housekeeping genes.

Tex domain architecture and sequence conservation may extend beyond prokaryotes to the essential eukaryotic transcription elongation factor Spt6.5, 6, 7 Tex is approximately half the size of Spt6 (e.g., 86 kDa for P. aeruginosa Tex versus 168 kDa for Saccharomyces cerevisiae Spt6), with sequence homology spanning the central region of Spt6. The flanking nonhomologous regions of Spt6 include a highly charged N-terminal region and a C-terminal SH2-like domain. Within the region of homology, Tex and Spt6 share ∼ 25% pairwise sequence identity and have a similar predicted domain architecture; primary sequence analysis identified YqgF, HhH, and S1 RNA-binding domains in both proteins.7 This level of sequence similarity falls in Doolittle's “twilight zone,”8 indicating that Tex and Spt6 may have similar structures, although direct evidence is lacking.

The sequence similarity may also indicate that Tex and Spt6 have related cellular functions. Although current evidence suggests that Spt6 is a nucleosome chaperone,9, 10, 11 a function unique to eukaryotes, recent studies have shown that Spt6 also interacts directly with both RNA polymerase (RNAP)12 and mRNA processing factors, including the exosomal RNA degradation machinery.13 Thus, beyond its role in nucleosome maintenance, Spt6 appears to provide a physical link between transcription and pre-mRNA surveillance, although the relationship between these critical processes is lacking in structural detail. Interestingly, we have recently observed similar interactions with Tex. P. aeruginosa Tex copurifies with RNAP, RNase E, and PNPase (I.V.-G. and S.L.D., unpublished data); RNase E and PNPase are components of the prokaryotic RNA degradosome, a 3′–5′ RNA degradation complex analogous to the eukaryotic exosome.14

In an effort to better understand the molecular function of Tex, and possibly to gain insight into Spt6, we have determined high-resolution crystal structures of the P. aeruginosa Tex protein in two crystal forms. These reveal four putative nucleic acid binding/modifying domains including a helix–turn–helix (HtH) domain that was not predicted from primary sequence. In addition, we have quantitatively examined the ability of Tex to bind various nucleic acid substrates and have found that Tex has a strong preference for single-stranded RNA (ssRNA). Binding appears to be sequence nonspecific, and mutagenesis studies indicate that this interaction is mediated by the flexible S1 domain. In contrast to an earlier proposal,1, 2 we do not observe significant nuclease function associated with the Tex YqgF domain. Our findings provide a structural foundation for understanding Tex function and can guide future studies on the structure and function of Spt6.

Section snippets

Structure determination and overall description

The full-length P. aeruginosa Tex protein was expressed recombinantly in Escherichia coli and purified by Ni-chelate, heparin affinity, and gel filtration chromatography. The C-terminal hexahistidine tag was retained for the structural and biochemical studies. The structure was determined by single-wavelength anomalous dispersion phasing and density modification using data collected to 2.7 Å resolution from a selenomethionine (Se-Met)-substituted crystal. This structure was refined against data

Tex protein expression and purification

Full-length Tex from P. aeruginosa strain PAO1 was cloned into a pET24 kan expression vector containing a C-terminal hexahistidine tag. The plasmid was transformed into cells of E. coli BL21-codonplus-(DE3)-RP (Stratagene). Cells were grown in LB media and induced with 1 mg/ml IPTG at an OD600 of 0.6 or alternatively grown using an autoinduction method as described in Ref. 41. In both cases, cells were grown at 37 °C for 5 h and then transferred to 20 °C and grown to saturation. Harvested cells

Acknowledgements

We thank Brenda Bass and members of the Bass Lab for use of materials and reagents as well as help with binding studies. This work was supported by National Institutes of Health Grants GM074368 (S.J.J.), T32-GM008537 (D.C.), AI057754 (S.L.D.), and GM076242 (C.P.H.). Financial support for use of the NSLS comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the U.S. Department of Energy and from the National Center for Research Resources of

References (53)

  • T. Grune et al.

    Crystal structure and functional analysis of a nucleosome recognition module of the remodeling factor ISWI

    Mol. Cell

    (2003)
  • F.W. Studier

    Protein production by auto-induction in high density shaking cultures

    Protein Expr. Purif.

    (2005)
  • G.D. Van Duyne et al.

    Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin

    J. Mol. Biol.

    (1993)
  • Z. Otwinowski et al.

    Processing of X-ray diffraction data collected in oscillation mode

  • T.M. Fuchs et al.

    A new gene locus of Bordetella pertussis defines a novel family of prokaryotic transcriptional accessory proteins

    J. Bacteriol.

    (1996)
  • L. Aravind et al.

    Survey and summary: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories

    Nucleic Acids Res.

    (2000)
  • E. Potvin et al.

    In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets

    Environ. Microbiol.

    (2003)
  • V. Anantharaman et al.

    Comparative genomics and evolution of proteins involved in RNA metabolism

    Nucleic Acids Res.

    (2002)
  • C.D. Kaplan et al.

    Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster

    Genes Dev.

    (2000)
  • C.P. Ponting

    Novel domains and orthologues of eukaryotic transcription elongation factors

    Nucleic Acids Res.

    (2002)
  • R.F. Doolittle

    Urfs and Orfs: A Primer on How to Analyze Derived Amino Acid Sequences

    (1986)
  • A. Bortvin et al.

    Evidence that Spt6p controls chromatin structure by a direct interaction with histones

    Science

    (1996)
  • C.D. Kaplan et al.

    Transcription elongation factors repress transcription initiation from cryptic sites

    Science

    (2003)
  • S.M. Yoh et al.

    The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export

    Genes Dev.

    (2007)
  • E.D. Andrulis et al.

    The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila

    Nature

    (2002)
  • A.J. Carpousis

    The Escherichia coli RNA degradosome: structure, function and relationship in other ribonucleolytic multienzyme complexes

    Biochem. Soc. Trans.

    (2002)
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