letters Crystal structure of the bacterial conjugation repressor FinO Alexandru F. Ghetu 1 , Michael J. Gubbins 2 , Laura S. Frost 2 and J. N. Mark Glover 1 1 Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada. 2 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada. The conjugative transfer of F-like plasmids is repressed by FinO, an RNA binding protein. FinO interacts with the F-plas- mid encoded traJ mRNA and its antisense RNA, FinP, stabiliz- ing FinP against endonucleolytic degradation and facilitating sense–antisense RNA recognition. Here we present the 2.0 Å resolution X-ray crystal structure of FinO, lacking its flexible N-terminal extension. FinO adopts a novel, elongated, largely helical conformation. An N-terminal region, previously shown to contact RNA, forms a positively charged α-helix (helix 1) that protrudes 45 Å from the central core of FinO. A C-terminal region of FinO that is implicated in RNA interactions also extends out from the central body of the protein, adopting a helical conformation and packing against the base of the N-terminal helix. A highly positively charged patch on the sur- face of the FinO core may present another RNA binding sur- face. The results of an in vitro RNA duplexing assay demonstrate that the flexible N-terminal region of FinO plays a key role in FinP–traJ RNA recognition, and supports our pro- posal that this region and the N-terminus of helix 1 interact with and stabilize paired, complementary RNA loops in a kiss- ing complex. The 35 kb transfer ( tra) operon of the F-plasmid encodes the proteins responsible for conjugative transfer of this plasmid from host to recipient Escherichia coli cells 1 . Expression of the tra oper- on is repressed by the two component FinOP system that includes the 186 residue, basic protein FinO, and the ~79 nucleotide RNA, FinP 2 . FinP contains two stem-loop structures (SLI and SLII) and is complementary to the untranslated leader of traJ , which encodes the primary transcriptional activator of the tra operon 3 . FinP is thought to interact with traJ mRNA to occlude its ribo- some binding site, blocking traJ translation and thereby inhibit- ing transcription of the tra operon 4 . Unprotected FinP is rapidly degraded by the cellular endo- nuclease RNase E and is, therefore, ineffective in repressing traJ translation 5 . FinO, the second component of the inhibition sys- tem, protects FinP against degradation. It binds to FinP and steri- cally blocks access to the RNase E cleavage site 5 . FinO also binds to the complementary stem-loop structures in traJ mRNA and pro- motes duplex formation between FinP and traJ RNA in vitro 6–8 . We showed that amino acids 26–61 and the remaining C-ter- minal residues (62–186) of FinO constitute two independent RNA binding regions 9 . The C-terminal region is also important for conferring RNase E resistance to FinP 10 . Within this region, residues 62–174 comprise a proteolytically stable domain that requires the rest of the C-terminal region (residues 175–186), or the N-terminal RNA binding region, for it to interact with RNA 9 . Here we present the crystal structure of a fragment of FinO (residues 26–186) that contains both RNA recognition regions. Using this structure, we have built a testable model of a complex of FinO bound to RNA comprising a minimal binding sequence nature structural biology • volume 7 number 7 • july 2000 565 and suggest a means by which FinO could facilitate FinP–traJ RNA interactions. Structure determination FinO protein was overexpressed and purified from E. coli as described 9 . Attempts to crystallize full length FinO from the F- like plasmid R6-5 were unsuccessful. We have shown that FinO lacking only the N-terminal 25 amino acids (FinO(26–186)) comprises a proteolytically stable fragment at 4 °C that binds FinP RNA with the same affinity as the full length protein 9 . FinO(26–186) crystallized at 4 °C in the space group P2 1 2 1 2 1 (a = 37.57 Å, b = 38.73 Å, c = 145.42 Å) with one FinO molecule per asymmetric unit. The crystals diffracted X-rays weakly to 3.0 Å on a rotating anode X-ray source. The structure of FinO(26–186) was solved by multiple wave- length anomalous dispersion (MAD) methods using selenome- thionine substituted protein. Native FinO has only one methionine site at which selenomethionine can be introduced. To increase the phasing power of the data collected from selenomethionine substituted crystals, we mutated leucines 96 and 124 to methionines (see Methods). Crystals of the selenome- thionine substituted double mutant FinO(26–186)L96,124M had the same unit cell dimensions as the wild type. Electrophoretic gel mobility shift analysis demonstrated that FinO(26–186)L96,124M binds FinP RNA with the same affinity as wild type FinO and FinO(26–186) (data not shown). A three- wavelength MAD data set was collected at beamline X12C at the National Synchrotron Light Source (NSLS). Solvent flattened, MAD phased electron density maps calculated to 2.6 Å resolu- Fig. 1 Stereo view of electron density maps around residues 152–156 (on strand β4) with the final 2.0 Å refined model superimposed. a, Density modified, 2.6 Å resolution MAD phased experimental map contoured at 1.0σ. b, 2F o - F c map at 2.0 Å resolution using phases calculated from the final, refined model and contoured at 1.6σ. a b © 2000 Nature America Inc. • http://structbio.nature.com © 2000 Nature America Inc. • http://structbio.nature.com