Crystal Structure of the T4 regA Translational Regulator Protein at 1.9 A Resolution ChulHee Kang,*t Rodney Chan, Imre Berger, Curtis Lockshin, Louis Green, Larry Gold, Alexander Richt The translational regulator protein regA is encoded by the T4 bacteriophage and binds to a region of messenger RNA (mRNA) that includes the initiator codon. RegA is unusual in that it represses the translation of about 35 early T4 mRNAs but does not affect nearly 200 other mRNAs. The crystal structure of regA was determined at 1.9 A resolution; the protein was shown to have an a-helical core and two regions with antiparallel P sheets. One of these P sheets has four antiparallel strands and has some sequence homology to RNP-1 and RNP-2, which are believed to be RNA-binding motifs and are found in a number of known RNA-binding proteins. Structurally guided mutants may help to uncover the basis for this variety of RNA interaction. The three-dimensional (3D) structures of many DNA-binding proteins have been solved, and the mechanism of sequence- specific DNA recognition is largely under- stood. In contrast, little is known about the recognition systems of proteins that bind to RNA in a sequence-specific manner. Most information concerning such interactions has come from crystal structures of the tRNA aminoacyl synthetases bound to their cognate tRNA molecules (1). Many proteins that bind to RNAs and influence mRNA splicing or translation have charac- teristic short amino acid sequences that are believed to participate in RNA recognition (2). These proteins are present in eubacte- ria and eukaryotes, and they usually bind to only one or a few target RNAs. However, the bacteriophage T4 regA protein binds many early T4 mRNAs and diminishes translation by blocking ribosome move- ment (3). Phage T4 encodes nearly 300 proteins, and as many as 35 of the 200 early genes are regulated by the regA protein (3). In comparing binding sites of target mRNAs, it has not been possible to identify a consensus sequence (3, 4). How does a protein with 122 amino acids translation- ally repress some 35 different mRNAs while ignoring the other mRNAs that are found in an infected cell at the same time? Here, we present the 3D structure of the regA protein and show that it shares some fea- tures with other proteins that are known to be important in binding to RNA. The expression, purification (5), crystal- lization, and structure determination of the regA protein are described in Tables 1 and 2. The crystals diffracted to better than 1.9 0 A resolution and belong to the orthorhom- bic space group P212121 with two molecules in the asymmetric unit (Table 1). The a carbon positions of the two molecules in the asymmetric unit are shown in Fig. 1. The molecule has three large cx-helical seg- ments and one turn of a 310 helix (Fig. 2). There are two regions with P pleated sheets: Sheet A contains three antiparallel strands and one parallel strand and sheet B con- tains four antiparallel strands. In the crystal structure, two identical polypeptide chains are brought together to form a dimer (Fig. 1) with a noncrystallographic pseudo-two- fold axis. The dimer interface is stabilized by a symmetrical pair of intersubunit salt bridges between Arg91 and Glu68 as well as by a symmetrical pair of intersubunit back- bone hydrogen bonds between the carbonyl group of Thr92 of one molecule and the amino group of the corresponding Thr92 of the other molecule. These interactions sug- gest the possibility that the molecule may exist as a dimer when it is biologically ac- tive; in fact, the molecule exists as a dimer in dilute solution (6). Several RNA-bind- ing proteins are known to exist as dimers, including the viral MS2 coat protein (7). The main structural differences between the two regA molecules in the asymmetric unit are found near residues 95 to 100 (Fig. 1) and at the COOH-terminal region (res- idues 119 to 122). The two COOH-termi- nal residues are disordered in one molecule. The root mean square (rms) deviation be- tween the positions of the at carbons of the two molecules is 1.2 A. When residues 95 to 100 and 119 to 122 are taken out, the rms deviation drops to 0.6 A. P sheet structures appear to be important components in RNA recognition. In the crystal structure of glutaminyl-tRNA com- plexed to its tRNA synthetase, a P pleated C. Kang, R. Chan, C. Lockshin, A. Rich, Department of Biology, Massachusetts Institute of Technology, Cam- bridge, MA 02139, USA. 1. Berger, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, and De- partment of Biophysical Chemistry, Hannover Medical School, 30623 Hannover, Germany. L. Green, Nexagen Inc., 2869 Wilderness Place, Boulder, CO 80302, USA. L. Gold, Department of Molecular, Cellular and Develop- mental Biology, University of Colorado, Boulder, CO 80302, USA. *Present address: Department of Biochemistry and Bio- physics, Washington State University, Pullman, WA 99164, USA. tTo whom correspondence should be addressed. 1170 Fig. 1. Stereo diagram illustrating the position of at carbon atoms in the two regA molecules found in the asymmetric unit. The last two COOH-terminal residues in the upper molecule are disordered. The molecules are organized around a noncrystallographic pseudo-twofold axis perpendicular to the page. SCIENCE * VOL. 268 * 26 MAY 1995 aiwag~ 1 Rill: 1.1m, 1:.,RWPW*Iwww#q-~ on February 17, 2015 www.sciencemag.org Downloaded from on February 17, 2015 www.sciencemag.org Downloaded from on February 17, 2015 www.sciencemag.org Downloaded from on February 17, 2015 www.sciencemag.org Downloaded from