Binding of complement subcomponent C1q to Streptococcus pyogenes: evidence for interactions with the M5 and FcRA76 proteins

Binding of C1q, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of C1q to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between C1q and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound C1q; alkaline and SDS extracts yielded the 52 kDa component as the main C1q-binding substance. Trypsin treatment of the SDS extracts of two GAS strains suggested the C1q-binding component(s) to be of protein nature. C1q-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human C1q and fibrinogen, conjugated to horse radish peroxidase (HRP) as well as rabbit IgG antibodies complexed to HRP (PAP system). The 52 kDa component was non-reactive with fibrinogen or rabbit IgG. However, C1q-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either fibrinogen (type M5) or both fibrinogen and rabbit IgG (type M76); the molecular mass of these components, 55 kDa and 43–40 kDa, respectively, was in agreement with the reported molecular mass of the M5 and FcRA76 proteins. Our findings suggest that C1q may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with C1q as biologically important.     

Insertional inactivation of virR in Streptococcus pyogenes M49 demonstrates that VirR functions as a positive regulator of ScpA, FcRA, OF, and M protein

Abstract Several reports have shown that Streptococcus pyogenes strains which produce opacity factor (OF+) have diverged significantly from OF⁻ serotypes. This study questions whether several surface proteins of an OF+ culture are regulated by the positive regulatory protein VirR, in a manner similar to OF~ strains. Interruption of the virR region of an OF⁺ S. pyogenes (strain CS101, M type 49) was performed using a temperature-sensitive plasmid containing a fragment of virR . Interruption of the virR region produced cultures with (indétectable amounts of M49 and ScpA proteins, and reduced the yield of FcRA protein. In addition, mutants had a significant reduction in detectable opacity factor. These results suggest that virR functions as a positive regulator of a variety of surface proteins in OF⁺ strains.     

DRBP76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR.

The interferon-induced double-stranded RNA-activated protein kinase PKR is the prototype of a class of double-stranded (dsRNA)-binding proteins (DRBPs) which share a dsRNA-binding motif conserved from Drosophila to humans. Here we report the purification of DRBP76, a new human member of this class of proteins. Sequence from the amino terminus of DRBP76 matched that of the M phase-specific protein, MPP4. DRBP76 was also cloned by the yeast two-hybrid screening of a cDNA library using a mutant PKR as bait. Analysis of the cDNA sequence revealed that it is the full-length version of MPP4, has a bipartite nuclear localization signal, two motifs that can mediate interactions with both dsRNA and PKR, five epitopes for potential M phase-specific phosphorylation, two potential sites for phosphorylation by cyclin-dependent kinases, a RG2 motif present in many RNA-binding proteins and predicts a protein of 76 kDa. DsRNA and PKR interactions of DRBP76 were confirmed by analysis of in vitro translated and purified native proteins. Cellular expression of an epitope-tagged DRBP76 demonstrated its nuclear localization, and its co-immunoprecipitation with PKR demonstrated that the two proteins interact in vivo. Finally, purified DRBP76 was shown to be a substrate of PKR in vitro, indicating that this protein's cellular activities may be regulated by PKR-mediated phosphorylation.     

Matrix genes of measles virus and canine distemper virus: cloning, nucleotide sequences, and deduced amino acid sequences.

The nucleotide sequences encoding the matrix (M) proteins of measles virus (MV) and canine distemper virus (CDV) were determined from cDNA clones containing these genes in their entirety. In both cases, single open reading frames specifying basic proteins of 335 amino acid residues were predicted from the nucleotide sequences. Both viral messages were composed of approximately 1,450 nucleotides and contained 400 nucleotides of presumptive noncoding sequences at their respective 3' ends. MV and CDV M-protein-coding regions were 67% homologous at the nucleotide level and 76% homologous at the amino acid level. Only chance homology was observed in the 400-nucleotide trailer sequences. Comparisons of the M protein sequences of MV and CDV with the sequence reported for Sendai virus (B. M. Blumberg, K. Rose, M. G. Simona, L. Roux, C. Giorgi, and D. Kolakofsky, J. Virol. 52:656-663; Y. Hidaka, T. Kanda, K. Iwasaki, A. Nomoto, T. Shioda, and H. Shibuta, Nucleic Acids Res. 12:7965-7973) indicated the greatest homology among these M proteins in the carboxyterminal third of the molecule. Secondary-structure analyses of this shared region indicated a structurally conserved, hydrophobic sequence which possibly interacted with the lipid bilayer.     

A role for the fibrinogen-binding regions of streptococcal M proteins in phagocytosis resistance

All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type-specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen-binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen-binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.     

Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions

Biologically active proteins without stable ordered structure (i.e., intrinsically disordered proteins) are attracting increased attention. Functional repertoires of ordered and disordered proteins are very different, and the ability to differentiate whether a given function is associated with intrinsic disorder or with a well-folded protein is crucial for modern protein science. However, there is a large gap between the number of proteins experimentally confirmed to be disordered and their actual number in nature. As a result, studies of functional properties of confirmed disordered proteins, while helpful in revealing the functional diversity of protein disorder, provide only a limited view. To overcome this problem, a bioinformatics approach for comprehensive study of functional roles of protein disorder was proposed in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Applying this novel approach to Swiss-Prot sequences and functional keywords, we found over 238 and 302 keywords to be strongly positively or negatively correlated, respectively, with long intrinsically disordered regions. This paper describes approximately 90 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions.     

Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins

Currently, the understanding of the relationships between function, amino acid sequence, and protein structure continues to represent one of the major challenges of the modern protein science. As many as 50% of eukaryotic proteins are likely to contain functionally important long disordered regions. Many proteins are wholly disordered but still possess numerous biologically important functions. However, the number of experimentally confirmed disordered proteins with known biological functions is substantially smaller than their actual number in nature. Therefore, there is a crucial need for novel bionformatics approaches that allow projection of the current knowledge from a few experimentally verified examples to much larger groups of known and potential proteins. The elaboration of a bioinformatics tool for the analysis of functional diversity of intrinsically disordered proteins and application of this data mining tool to >200 000 proteins from the Swiss-Prot database, each annotated with at least one of the 875 functional keywords, was described in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V.N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Using this tool, we have found that out of the 710 Swiss-Prot functional keywords associated with at least 20 proteins, 262 were strongly positively correlated with long intrinsically disordered regions, and 302 were strongly negatively correlated. Illustrative examples of functional disorder or order were found for the vast majority of keywords showing strongest positive or negative correlation with intrinsic disorder, respectively. Some 80 Swiss-Prot keywords associated with disorder- and order-driven biological processes and protein functions were described in the first paper (see above). The second paper of the series was devoted to the presentation of 87 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions (Vucetic, S.; Xie, H.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions. J. Proteome Res. 2007, 5, 1899-1916). Protein structure and functionality can be modulated by various post-translational modifications or/and as a result of binding of specific ligands. Numerous human diseases are associated with protein misfolding/misassembly/misfunctioning. This work concludes the series of papers dedicated to the functional anthology of intrinsic disorder and describes approximately 80 Swiss-Prot functional keywords that are related to ligands, post-translational modifications, and diseases possessing strong positive or negative correlation with the predicted long disordered regions in proteins.     

Properties of isolated human alpha1-antitrypsins of Pi types M, S and Z.

1. alpha1-Antitrypsin contains a single thiol group partly blocked in native plasma and reactive after mild reduction. 2. Human alpha1-antitrypsins of Pi types F, M, S and Z have been isolated with native microheterogeneity using thiol-disulfide (SH-SS) interchange reactions utilizing the reactive thiol group. 3. The pI of the various microheterogeneous fractions are given for protein M. Stepwise desialylation of alpha1-antitrypsin indicates that the charge difference between the major fractions is one sialic acid residue between each. This is further supported by the pI changes obtained on substitution of the single thiol with positively or negatively charged compounds. 4. Desialyation of purified proteins from each Pi type converts the individual microheterogeneous fractions to one major fraction. The pI shift for the variants studied indicate a difference of plus or minus one or two charge units between protein M and the variants. 5. A difference of one sialic acid residue was obtained for proteins M and Z by the thiobarbituric assay, but stepwise removal of sialic acid with neuraminidase revealed almost identical stepwise change of pattern of both proteins indicating the same number of sialic acid residues. 6. Electrofocusing has been used to identify CNBr fragments from proteins M, S and Z. 7. An amino acid substitution has been found to be located in one of the eight CNBr fragments, glutamic acid in protein M is substituted by lysine in protein Z. 8. The average concentration of alpha1-antityprsin in plasma from healthy males was found to be 1.32 g/1.     

Complete amino acid sequence of streptococcal PepM49 protein, a nephritis-associated serotype. Conserved conformational design among sequentially distinct M protein serotypes

The complete amino acid sequence of PepM49, a peptic fragment of the group A streptococcal type 49 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment retains the opsonic antibody epitope of the native molecule. The sequence of PepM49, as determined by automated Edman degradations of the uncleaved molecule, and its tryptic and chymotryptic peptides, consists of a total of 143 residues (Mr = 17,187). PepM49, a nephritis-associated M protein serotype, exhibits significant internal homology in its sequence. However, identical sequence repeats of the kind seen in the rheumatic fever-associated serotypes M5, M6, and M24, are absent in PepM49. PepM49 exhibits varying degrees of homology with the M5, M6, and M24 proteins, which is consistent with the existence of variable and conserved regions in the M protein molecule. Predictive analysis as well as CD measurements revealed a high propensity of the PepM49 molecule to assume an alpha-helical conformation. Furthermore, a heptad periodicity of the nonpolar residues, a characteristic of alpha-helical coiled-coil proteins, extends over the entire length of the PepM49 protein. The differences in the nonpolar residue distribution divide the PepM49 sequence into three distinct domains, similar to those seen earlier in the M5 and M6 proteins. Together, these studies establish a conserved conformational design for the sequentially diverse M protein serotypes. However, the pattern of heptad periodicity in the PepM49 protein is quite distinct from that present in the PepM5 and M6 proteins, suggesting distinct differences in structural features among conformationally similar M protein serotypes. This may have relevance to the pathological differences associated with these M protein serotypes.     

Interactions between M protein and other structural proteins of severe,acute respiratory syndrome-associated coronavirus

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) structural proteins (S, E, M, and NC) localize in different subcellular positions when expressed individually. However, SARS-CoV M protein is co-localized almost entirely with S, E, or NC protein when co-expressed in the cells. On the other hand, only partial co-localization was observed when S and E, S and NC, or E and NC were co-expressed in the cells. Interactions between SARS-CoV M and other structural proteins but not interactions between S and E, S and NC, or E and NC were further demonstrated by co-immunoprecipitation assay. These results indicate that SARS-CoV M protein, similar to the M proteins of other coronaviruses, plays a pivotal role in virus assembly. The cytoplasmic C-terminus domain of SARS-CoV M protein was responsible for binding to NC protein. Multiple regions of M protein interacted with E and S proteins. A model for the interactions between SARS-CoV M protein and other structural proteins is proposed. This study helps us better understand protein-protein interactions during viral assembly of SARS-CoV. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tritium planigraphy: Differences in the spatial structures of the influenza virus M1 protein in crystal,solution, and virion

The structure of the M1 protein of the influenza virus A/Puerto Rico/8/34 (PR8, subtype H1N1) in solution at acidic pH and in the composition of the virion has been studied by the tritium planigraphy method. A model of the spatial structure was constructed using a special algorithm simulating the experiment and a set of algorithms for predicting the secondary structure and disordered regions in proteins. The tertiary structure was refined using the Rosetta program. For a comparison of the structures in solution and inside the virion, the data of X-ray diffraction analysis for the NM domain were also used. The main difference in the structures of the protein in solution and the crystalline state is observed in the region of contact of N and M domains, which in the crystalline state is packed more densely. The regions of the maximum label incorporation almost completely coincide with unstructured regions in the protein that were predicted by the bioinformatics analysis. These regions are concentrated in the C domain and in loop regions between M, N, and C domains. The data were confirmed by analytical centrifugation and dynamic light scattering. Anomalous hydrodynamic dimensions and a low structuration of the M1 protein in solution were found. The polyfunctionality of the protein in the cell is probably related to its flexible tertiary structure, which, owing to unstructured regions, provides contact with various partner molecules.     

Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.

Biochemical and pharmacological data support the existence of multiple forms of the Na/H exchanger (NHE). Two isoforms, termed NHE-1 and NHE-2, have recently been isolated from rabbit ileal villus epithelial cells (Tse, C. M., Ma, A. I., Yang, V. W., Watson, A. J. M., Levine, S., Montrose, M. H., Potter, J., Sardet, C., Pouysségur, J., and Donowitz, M. (1991) EMBO J. 10, 1957-1967; Tse, C. M., Watson, A. J. M., Ma, A. I., Pouysségur, J., and Donowitz, M. (1991) Gastroenterology 100, A258). To identify additional molecular forms of the exchanger, rat brain, heart, kidney, stomach, and spleen cDNA libraries were screened for their presence using an NHE-1 cDNA probe under low stringency hybridization conditions. cDNAs encoding rat NHE-1 and two structurally related proteins, designated NHE-3 and NHE-4, have been isolated. Based on the deduced amino acid sequences, NHE-1, -3, and -4 are similar in size, having relative molecular masses of 91,506, 92,997, and 81,427, respectively. Overall, the proteins exhibit approximately 40% amino acid identity to each other and have similar hydropathy profiles, suggesting that they have the same transmembrane organization. The predicted N-terminal transmembrane regions of the three proteins, which span between 453 and 503 amino acids, exhibit the highest degree of identity (45-49%). In contrast, the C-terminal cytoplasmic regions, which span between 247 and 378 amino acids, exhibit very low amino acid identity (24-31%). Tissue distribution studies reveal that the NHE-1 mRNA is present at varying levels in all tissues examined, whereas NHE-3 and NHE-4 mRNAs exhibit a more limited distribution. NHE-3 mRNA is expressed at high levels in colon and small intestine, with significant levels also present in kidney and stomach. NHE-4 mRNA is most abundant in stomach, followed by intermediate levels in small intestine and colon and lesser amounts in kidney, brain, uterus, and skeletal muscle. These data suggest that the molecular basis for the functional diversity of the Na/H exchanger in mammals is based, at least in part, on expression of multiple members of a gene family.     

Domain structure and molecular flexibility of streptococcal M proteinIn Situ probed by limited proteolysis

Serologically distinct group A streptococcal M proteins, the antiphagocytic determinants of the bacteria, have a highly repetitive sequence and exhibit a heptad periodicity characteristic of alpha-helical coiled-coil proteins. Based on the differences in the pattern of heptad periodicity, the coiled-coil region of the complete M molecule has been divided into three distinct domains: I, II, and III. Domains I and II together constitute the variable part of M protein, whereas domain III is conserved among serotypes. Pepsin treatment of the M5, M6, and M24 streptococci results in a preferential cleavage of their M molecules between the predicted domains II and III, releasing biologically active fragments of the respective M proteins. Thus, a pepsin cleavage site at the junction of their variable and conserved regions is conserved in the M5, M6, and M24 proteins. In contrast, in the case of the M49 streptococci, the primary site of pepsin cleavage was observed to be within the conserved region of the M49 molecule, rather than at the junction of its variable and conserved regions. Despite containing part of the conserved region, the PepM49 protein is significantly smaller than the pepsin fragments of the M5, M6, and M24 proteins, which contain only the variable regions. However, in addition to the major PepM49 species, the pepsin digest of the type-49 streptococci also contained a smaller fragment, PepM49/a, as a minor component. Its formation was extremely sensitive to thepH of pepsin digestion. PepM49/a, which retains both the propensity to attain an alpha-helical conformation and the opsonic antibody epitope of the M49 molecule, contains only domains I and II like the other PepM proteins. Thus, as in the M5, M6, and M24 proteins, a pepsin cleavage site at the junction of the variable and conserved regions is indeed present in the M49 molecule, but is much less accessible relative to the other serotypes. Thus, the pepsin cleavage sites in the M protein correlate quite well with the boundaries of structurally distinct domains reflected by the predictive analysis. These sites apparently represent the flexible/hinge regions of the molecule. PepM49/a is the least repetitive and the shortest of the M protein pepsin fragments isolated so far. These results suggest that the flexibility of the interdomain regions in M protein may be dependent on the molecular size of their variable domains. The placement of a more accessible hinge within the conserved part of the M49 molecule, rather than at the junction of the variable and conserved domains, suggests that a critical molecular size may be essential for the efficient functioning of the M molecule.     

Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M.

Oncostatin M is a polypeptide of Mr approximately 28,000 that acts as a growth regulator for many cultured mammalian cells. We report the cDNA and genomic cloning, sequence analysis, and functional expression in heterologous cells of oncostatin M. cDNA clones were isolated from mRNA of U937 cells that had been induced to differentiate into macrophagelike cells by treatment with phorbol 12-myristate 13-acetate, and a genomic clone was also isolated from human brain DNA. Sequence analysis of these clones established the 1,814-base-pair cDNA sequence as well as exon boundaries. This sequence predicted that oncostatin M is synthesized as a 252-amino-acid polypeptide, with a 25-residue hydrophobic sequence resembling a signal peptide at the N terminus. The predicted oncostatin M amino acid sequence shared no homology with other known proteins, but the sequence of the 3' noncoding region of the cDNA contained an A + T-rich stretch with sequence motifs found in the 3' untranslated regions of many cytokine and lymphokine cDNAs. Oncostatin M mRNA of approximately 2 kilobase pairs was detected in phorbol 12-myristate 13-acetate-treated U937 cells and in activated human T cells. Transfection of cDNA encoding the oncostatin M precursor into COS cells resulted in the secretion of proteins with the structural and functional properties of oncostatin M. The unique amino acid sequence, expression by lymphoid cells, and growth-regulatory activities of oncostatin M suggest that it is a novel cytokine.     

Domains of group A streptococcal M protein that confer resistance to phagocytosis, opsonization and protection: implications for vaccine development

Streptococcus pyogenes (group A streptococcus) colonizes skin and throat tissues resulting in a range of benign and serious human diseases. Opsonization and phagocytosis are important defence mechanisms employed by the host to destroy group A streptococci. Antisera against the cell-surface M protein, of which over 150 different types have been identified, are opsonic and contribute to disease protection. In this issue of Molecular Microbiology, Sandin and colleagues have comprehensively analysed the regions of M5 protein that contribute to phagocytosis resistance and opsonization. Human plasma proteins bound to M5 protein B- and C-repeats were shown to block opsonization, an observation that needs to be carefully considered for the development of M protein-derived vaccines. While safe and efficacious human group A streptococcal vaccines are not commercially available, candidate M protein-derived vaccines have shown promise in murine vaccine models and a recent phase 1 human clinical trial.     

Sequential binding of DNA repair proteins RPA and ERCC1 to XPA in vitro.

Recent studies have shown that many proteins are involved in the early steps of nucleotide excision repair and that there are some interactions between nucleotide excision repair proteins, suggesting that these interactions are important in the reaction mechanism. The xeroderma pigmentosum group A protein (XPA) was shown to bind to the replication protein A (RPA) or the excision repair cross complementing rodent repair deficiency group 1 protein (ERCC1), and these interactions might be involved in the damage-recognition and/or incision steps, of nucleotide excision repair. Here we show that the XPA regions required for the binding to the 70 and 34 kDa subunits of RPA are located in the middle and on N-terminal regions of XPA, respectively. These regions do not overlap with the ERCC1-binding region of XPA, and a ternary protein complex of RPA, XPA and ERCC1 was detected in vitro. In addition, using the surface plasmon resonance biosensor, the binding of RPA and ERCC1 to XPA was investigated. The dissociation constants (KD) of RPA and ERCC1 with XPA were 1.9 x 10(-8 )and 2.5 x 10(-7) M, respectively. Moreover, our results suggest the sequential binding of RPA and ERCC1 to XPA.     

Sequence homology of group A streptococcal Pep M5 protein with other coiled-coil proteins

Group A streptococcal Pep M5 protein, an antiphagocytic determinant of the bacteria, is an alpha-helical coiled-coil molecule, and exhibits significant sequence homology with tropomyosin and myosin, but to a lesser degree with other coiled-coil proteins. Moreover, Pep M5 is more homologous to myosin than to tropomyosin, and the homologies are more numerous between the C-terminal domain of the Pep M5 protein and the S2 fragment of myosin. The C-terminal domain of the Pep M5 protein exhibits extensive sequence identity with the C-terminal region of Pep M6 molecule, another M protein serotype. Thus, regions within two M protein serotypes are homologous to the S2 region of the myosin molecule. These observations are consistent with the immunological findings of other investigators and thus may explain some of the previously reported immunological cross-reactions between antigens of the group A streptococcus and mammalian heart tissue.     

Salt dissociation of nuclear particles containing DNA-like RNA. Distribution of phosphorylated and nonphosphorylated species.

Electrophoresis of proteins from nuclear particles containing DNA-like RNA gave a pattern with 45 bands. The possibility that some of these proteins arose by contamination with ribosomes, chromatin, or soluble nuclear proteins was examined and eliminated. The fate of the proteins of the particles was studied after partial dissociation with 0.25 and 0.70 M NaCl. The individual proteins were released progressively and in different quantities. A group of easily released species (75 and 95% removed with 0.25 and 0.70 M NaCl) was demonstrated. This group contained 8 species between 29,000 and 39,000 daltons which represented approximately one-half of the total number of molecules. It is suggested that they are bound to repetitive sequences of the RNA. At least 30 and 60% of the other proteins were released at 0.25 and 0.70 M NaCl, respectively. There were no specific proteins tightly bound to the RNA, unless the nature of the remaining species is different from that of the released ones of the same molecular weight. The phosphorylated proteins were more tightly bound to the RNA than the nonphosphorylated species of similar molecular weight. In several instances, the 32-P radioactivity was associated with quantitatively minor bands of proteins.