The C proteins (Cl and C2) of HeLa 40S heterogeneous nuclear ribonucleoprotein particles copurify under native conditions as a stable complex with a fixed molar protein ratio (S. F. Barnett, W. M. LeStourgeon, and D. L. Friedman, J. Biochem. Biophys. Methods 16:87-97, 1988). Gel filtration chromatography and velocity sedimentation analyses of these complexes revealed a large Stokes radius (6.2 nm) and a sedimentation coefficient of 5.8S. On the basis of these values and a partial specific volume of 0.70 cm3/g based on the amino acid composition, the molecular weight of the complex was calculated to be 135,500. This corresponds well to 129,056, the sequence-determined molecular weight of a (Cl)3C2 tetramer. Reversible chemical cross-linking with dithiobis(succinimidyl propionate) and analysis of cross-linked and cleaved complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the C proteins exist as tetramers, most or all of which are composed of (C1)3C2. The tetramer is stable in a wide range of NaCl concentrations (0.09 to 2.0 M) and is not dissociated by 0.5% sodium deoxycholate. This stability is not the result of disulfide bonds or interactions with divalent cations. The hydrodynamic properties of highly purified C-protein tetramers are the same for C-protein complexes released from intact particles with RNase or high salt. These findings support previous studies indicating that the core particle protein stoichiometry of 40S heterogeneous nuclear ribonucleoproteins is N(3A1-3A2-1B1-1B2-3C1-1C2), where N = 3 to 4, and demonstrate that the C-protein tetramer is a fundamental structural element in these RNA-packaging complexes. The presence of at least three tetramers per 40S monoparticle, together with the highly anisotropic nature of the tetramer, suggests that one-third of the 700-nucleotide pre-mRNA moiety packaged in monoparticles is associated through a sequence-independent mechanism with the C proteins.In mammalian cells, pre-mRNA is packaged during transcription by multiple copies of a few abundant nuclear proteins to form a repeating array of regular 40S heterogeneous nuclear ribonucleoprotein particles (hnRNP) (11, 13, 15, 22; W. M. LeStourgeon, S. F. Barnett, and S. J. Northington, in S. H. Wilson, ed., The eukaryotic nucleus, in press). When isolated from sucrose density gradients, 40S monoparticles possess an ultrastructural morphology indistinguishable from that seen in gently spread transcriptive units (38) and are composed primarily of multiple copies of six major core particle proteins (5,7,14,24,25,27,40) and a 700-nucleotide fragment of RNA (13). The proteins migrate in single-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as three groups of doublet bands (A1-A2, B1-B2, and C1-C2 doublets) with apparent molecular masses of 32 to 44 kilodaltons (5). Studies with protein-cross-linking reagents have indicated that the basic molar ratio of the core proteins in particles from rapidly dividing HeLa cells is 3A1-3A2-1B1-1B2-3C1-1C2 (27). W...