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LicT is a bacterial regulatory protein able to prevent the premature arrest of transcription. When activated, LicT binds to a 29 base RNA hairpin overlapping a terminator located in the 5' mRNA leader region of the target genes. We have determined the solution structure of the LicT RNA-binding domain (CAT) in complex with its ribonucleic antiterminator (RAT) target by NMR spectroscopy (PDB 1L1C). CAT is a beta-stranded homodimer that undergoes no important conformational changes upon complex formation. It interacts, through mostly hydrophobic and stacking interactions, with the distorted minor groove of the hairpin stem that is interrupted by two asymmetric internal loops. Although different in sequence, these loops share sufficient structural analogy to be recognized similarly by symmetry-related elements of the protein dimer, leading to a quasi- symmetric structure reminiscent of that observed with dimeric transcription regulators bound to palindromic DNA. Sequence analysis suggests that this RNA- binding mode, where the RAT strands are clamped by the CAT dimer, is conserved in homologous systems.

SacY is the prototype of a family of regulatory proteins able to prevent transcription termination. It interacts with a 29 nucleotide RNA sequence able to fold into a stem-loop structure and partially overlapping with a terminator sequence located in the 5' leader mRNA region of the gene it controls. We show here that the N-terminal fragment of SacY, SacY(1-55), and the corresponding fragments of other members of the family have antiterminator activities with efficiency and specificity identical to those of the full-length proteins. In vitro, this activity correlates with the specific affinity of SacY(1-55) for its RNA target. UV melting experiments demonstrate that SacY(1-55) binding stabilizes the RNA target structure. The NMR solution structure of SacY(1-55) is very similar to that obtained in the crystal (van Tilbeurgh et al., 1997): the peptide is folded as a symmetrical dimer without any structural homology with other RNA-binding domains yet characterized. According to a preliminary NMR analysis of the SacY(1-55)-RNA complex, the protein dimer is not disrupted upon RNA binding and several residues implicated in RNA recognition are located at the edge of the dimer interface. This suggests a new mode of protein-RNA interaction.

SacY belongs to a family of, at present, seven bacterial transcriptional antiterminators. The RNA-binding and antitermination capacity of SacY resides in the 55 amino acids at the N-terminal [SacY(1-55)]. The crystal structure at 2 A resolution shows that SacY(1-55) forms a dimer in the crystal, in accordance with the NMR solution structure. The structure of the monomer is a four-stranded beta-sheet with a simple beta1beta2beta3beta4 topology. One side of the sheet is covered by a long surface loop and the other side forms the dimer interface. The dimer is stabilized by the orthogonal stacking of the two beta-sheets. The crystal structure is in excellent agreement with the NMR solution structure (r.m.s. distance for C alpha coordinates is 1.3 A). The structure of SacY(1-55) reveals a new RNA-binding motif.