The Tex (t
pression) 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.1-3
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 function(s) of Tex remains enigmatic. Insight into Tex function is derived from several bacterial studies. Aside from its role in expression of toxin gene products in B. pertussis
gene from Pseudomonas 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 effect 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 Spt6.5-7
Tex is approximately half the size of Spt6 (e.g. 86 kDa for P. aeruginosa
Tex vs. 168 kDa for S. cerevisiae
Spt6), with sequence homology spanning the central region of Spt6. The flanking non-homologous 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,9-11
a function unique to eukaryotes, recent studies have shown that Spt6 also interacts directly with both RNA polymerase12
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 co-purifies with RNA polymerase (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. aeurginosa
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. Binding appears to be sequence non-specific 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.