We have determined the tertiary structure of box 2 from hamster HMG1 using bacterial expression and 3D NMR. The all a-helical fold is in the form of a V-shaped arrowhead with helices along two edges and one rather flat face. This architecture is not related to any of the known DNA binding motifs. Inspection of the fold shows that the majority of conserved residue positions in the HMG box family are those involved in maintaining the tertiary structure and thus all homologous HMG boxes probably have essentially the same fold. Knowledge of the tertiary structure permits an interpretation of the mutations in HMG boxes known to abrogate DNA binding and suggests a mode of interaction with bent and 4-way junction DNA.

The polypyrimidine tract binding protein (PTB) is an important regulator of alternative splicing that also affects mRNA localization, stabilization, polyadenylation, and translation. NMR structural analysis of the N-terminal half of PTB (residues 55-301) shows a canonical structure for RRM1 but reveals novel extensions to the beta strands and C terminus of RRM2 that significantly modify the beta sheet RNA binding surface. Although PTB contains four RNA recognition motifs (RRMs), it is widely held that only RRMs 3 and 4 are involved in RNA binding and that RRM2 mediates homodimerization. However, we show here not only that the RRMs 1 and 2 contribute substantially to RNA binding but also that full-length PTB is monomeric, with an elongated structure determined by X-ray solution scattering that is consistent with a linear arrangement of the constituent RRMs. These new insights into the structure and RNA binding properties of PTB suggest revised models of its mechanism of action.

The La protein is an important component of ribonucleoprotein complexes that acts mainly as an RNA chaperone to facilitate correct processing and maturation of RNA polymerase III transcripts, but can also stimulate translation initiation. We report here the structure of the C-terminal domain of human La, which comprises an atypical RNA recognition motif (La225-334) and a long unstructured C-terminal tail. The central beta sheet of La225-334 reveals novel features: the putative RNA binding surface is formed by a five-stranded beta sheet and, strikingly, is largely obscured by a long C-terminal alpha helix that encompasses a recently identified nuclear retention element. Contrary to previous observations, we find that the La protein does not contain a dimerization domain.

The HMGI-C protein is a nuclear phosphoprotein expressed at high levels in transformed cells. The cDNA encoding the mouse protein has been isolated and the sequence of the encoded protein shows that it is related to the HMGY and I proteins, proteins which bind in the minor groove of DNA containing stretches of A and T. The HMGI-C protein has three short highly basic domains, an acidic C-terminal domain, and potential CDC2/p34 and casein kinase II phosphorylation sites. Analysis of mRNA levels demonstrate that the HMGI-C gene is not expressed in a variety of mouse tissues but is expressed in Lewis lung carcinoma cells.

The binding of histones in chromatin core particles and in core particles depleted of histones H2A and H2B has been studied by high-resolution proton nuclear magnetic resonance (NMR) at 270 MHz. At low ionic strengths it is shown that histones H3 and H4 are bound in the core particle. Further, whereas the apolar regions of H2A and H2B are also bound to the core particle, the basic N-terminal and C-terminal regions are more mobile and give rise to sharp resonances in the NMR spectrum of the core particle. Between 0.3 and 0.6 M NaCl there is further release of basic regions of histones H3 and H4 from the complex. The dissociation of the core particle between 0.6 and 2.0 M NaCl is accompanied by the release of the structured apolar regions of the histones as evidenced by the appearance of a complex aromatic spectrum and perturbed upfield ring-currentshifted methyl resonances. Arginine residues are implicated in the binding between histones and DNA and 69'x of these residues are found in the apolar regions of the histones. Thc interactions between histones and DNA in the core particle thus involves H3 and H4 and the apolar regions of H2A and H2B. It is suggested that these basic regions of H2A and H2B have binding sites outside the core particle.

A simple procedure based on perchloric acid extraction has been developed for the preparation and purification of bovine prothymosin alpha and thymosins beta 4 and beta 9 in high yields. Spectroscopic observations show these proteins to be non-folding at neural pH. The cellular locations of human prothymosin alpha, rat parathymosin and calf thymosin beta 4, all so-called 'thymic hormones', have been studied by injection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that human prothymosin alpha and rat parathymosin both migrate to the nucleus whilst thymosin beta 4 remains in the cytoplasm. The peptide (1-88) of calf prothymosin alpha is shown not to accumulate in the Xenopus nucleus, demonstrating that the C-terminal 21 residues, which include a KKQK sequence, are required for nuclear migration. The present data, in association with existing evidence of wide tissue distribution and the lack of signal peptides, indicate that these proteins do not behave as hormones in the usual sense of the word. It is suggested that thymosin beta 4 should be grouped separately from the pro- and parathymosins.

Sox-5 is one of a family of genes which show homology to the HMG box region of the testis determining gene SRY. We have used indirect immunofluorescence to show that Sox-5 protein is localized to the nucleus of post-meiotic round spermatids in the mouse testis. In vitro footprinting and gel retardation assays demonstrate that Sox-5 binds specifically to the sequence AACAAT with moderately high affinity (Kd of approximately 10(-9) M). Moreover, interaction of Sox-5 with its target DNA induces a significant bend in the DNA, characteristic of HMG box proteins. Circular dichroism spectroscopy of the Sox-5 HMG box and its specific complex with DNA shows an alteration in the DNA spectrum, perhaps as a consequence of DNA bending, but none in the protein spectrum on complex formation. The dependence of the change in the CD spectrum with protein to DNA ratio demonstrates the formation of a 1:1 complex. Analysis of the structure of the Sox-5 HMG box by 2D NMR suggests that both the location of helical secondary structure as well as the tertiary structure is similar to that of HMG1 box 2.

High-resolution proton N M R spectrocopy has been used to study the solution structures of the subfragment 1 (SI) isoenzymes (containing either the A1 or A2 light chains) from rabbit skeletal muscle myosin and to investigate their interaction with actin. Superimposed upon broad components, the narrow signals of the S1 spectra are unexpectedly sharp, indicating that domains of varying sidechain mobility occur in the conformation adopted in solution. These observations are in agreement with previous studies of the mixed isoenzymes [Highsmith et al. (1 979) Biochemistry, 18, 4238 -42431. Peptide amide exchange studies show also that the S 1 structure accommodates fluctuations of sufficient amplitude to allow most of the peptide groups to come into contact with the solvent on the time scale-of the 'H-NMR experiment. The overall impression is that S1 is a molecule possessing backbone motility as well as domains of different sidechain mobility.Close comparison of the Sl(A1) and Sl(A2) spectra indicate that the N-terminal41 residues of the A1 light chain, rich in lysine, proline and alanine, display a high degree of segmental mobility. The difference spectrum [SI(AI)-Sl(A2)] obtained closely resembles the spectral simulation of the 41-residue segment. Upon addition of actin, many of the narrow S1 resonances decrease in intensity or progressively disappear altogether, indicative of intermediateslow exchange conditions consistent with the recognised high affinity between the two proteins. These changes are interpreted as an overall modulation in the observed and hence more mobile regions of S1 as has been suggested in earlier H-NMR studies referred to above. In particular, the differences noted between S l(A 1 ) and S l(A2) have now largely disappeared in their complexes with actin indicating a marked reduction in the segmental mobility of the N-terminal region of the light chain in S l(A 1). Together with other affinity chromatography results [Winstanley and Trayer (1 979) Biochem. Soc. Trans. 7, 703 -7041, this is good evidence for a direct interaction between this area of S l(A 1) and actin.The mechanism of muscle contraction, as originally proposed by Huxley [I] and modified by several groups of authors [2, 31, consists of a cycle of cross-bridge detachments and attachments, which are the basis of the 'sliding filament' theory, The myosin heads form cross-bridges with actin, which are then detached by ATP. The hydrolysis of ATP produces some conformational change in myosin which is restored as mechanical energy when the heads rebind actin. There is now ample evidence that this theory is correct. There are, however, considerable differences of opinion with regard to the intermolecular and intramolecular interactions involved in the actomyosin complex, and several models have been proposed, as reviewed by Taylor [4]. X-ray diffraction and electron microscopy indicate that the cross-bridge or part of it rotates during contraction [5] and the existence of a 'swivel' and a 'hinge' have been postulated in myosin [6 -1 I...