Analysis of protease-sensitive regions in the skeletal muscle sodium channel in vitro and implications for channel tertiary structure.

The tertiary structure of the rat skeletal muscle sodium channel was probed in vitro by determining regions of sensitivity to V-8 protease, trypsin, and chymotrypsin. Resultant channel fragments were identified with antibodies to defined sequences distributed along the primary structure. The temporal pattern of proteolysis was followed with channel protein in either detergent-phospholipid micelles or membrane fragments as well as with channel exposed to sodium dodecyl sulfate. Proteolysis in micelles and membranes occurred in discrete, reproducible steps that were similar in both systems. Although the size of intermediates varied slightly, their sequence of appearance was similar for all enzymes, suggesting that the observed pattern was determined by the relative accessibility of selected sites in the tertiary structure. No major change in channel organization appeared to occur after solubilization of membranes in nonionic detergents. Highly accessible sites in the native structure included the carboxyl terminus and the region linking the second and third internal repeat domains, while the amino terminus and the repeat domains themselves were relatively resistant to proteolysis unless the protein was denatured. Kinetically, interdomain II-III was the most readily cleaved; interdomains I-II and especially III-IV were less easily accessible. While domains I and IV appeared to remain intact throughout our experiments, limit fragments for epitopes associated with domains II and III suggest that cleavage eventually occurs at sites between the putative S5 and S6 helices in these domains.     

Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.     

Solution structures of the cytoplasmic linkers between segments S4 and S5 (S4-S5) in domains III and IV of human brain sodium channels in SDS micelles.

The two cytoplasmic linkers connecting segment S4 and segment S5 (S4-S5 linker) of both domain III (III/S4-S5) and IV (IV/S4-S5) of the sodium channel alpha-subunit are considered to work as a hydrophobic receptor for the inactivation particle because of the three hydrophobic amino acids of Ile-Phe-Met (IFM motif) in the III-IV linker of the sodium channel alpha-subunit. To date, the solution structures of the peptides related to III/S4-S5 (MP-D3: A1325-M1338) and IV/S4-S5 (MP-D4: T1648-L1666) of human brain sodium channels have been investigated using CD and (1)H NMR spectroscopies. SDS micelles were employed as a solvent. The micelles mimic either biological membranes or the interior of a protein and can be a relevant environment at the inactivated state of the channels. It was found that the secondary structures of both MP-D3 and MP-D4 assume alpha-helical conformations around the N-terminal half-side of the sequences, i.e. the residues between V1326 and L1331 in MP-D3 and between L1650 and S1656 in MP-D4. Residue A1329 in MP-D3, which is considered to interact with F1489 of the IFM motif, was found to be located within the alpha-helix. Residues F1651, M1654, M1655, L1657 and A1669 in MP-D4, which also play an important role in inactivation, formed a hydrophobic cluster on one side of the helix. This cluster was concluded to interact with the hydrophobic cluster due to the III-IV linker before the inactivation gate closes.     

Sodium Channel Fragments: Contributions to Voltage Sensitivity and Ion Selectivity

The peptide strategy was employed to resolve structure-function relationships in the voltage-dependent sodium channel. Two families of motifs were studied: the four voltage sensors S4 extended with the short cytoplasmic linkers L45 and the four P-regions, between S5 and S6, each from the homologous domains of the electric eel sodium channel. Macroscopic conductance experiments conducted with synthetic S4L45s in neutral lipid planar bilayers pointed to a moderate voltage-sensitivity for repeat IV which has no proline, whereas S4L45 of repeats I and II (Pro 19) and especially of repeat III (Pro 14) were much more voltage-sensitive. The influence both of Pro and its position within the sequence was confirmed by comparing the human skeletal muscle channel isoform D4/S4 wild-type and the R4P analogue. Circular dichroism spectroscopy shows highest and lowest helicities for repeats IV and III. The conformational transition (from helix to extended, mainly beta forms), which occurs when the solvent dielectric constant increases, was broader with repeat III. These structural and functional correlates suggest alternative gating mechanisms. The different contributions of each repeat also have effects at the level of the main selectivity filter, which suggests self-recognition between the four P-regions is a key component of intact sodium channel selectivity. In addition, the P-region from domain III is significantly voltage-sensitive and molecular dynamics simulations show that the C-terminal part of P-regions is mainly helical whilst the N-terminus tends to unfold. Such specializations of the four domains both in gating and selectivity are independently confirmed in recent electrophysiological studies.     

Unfolding-refolding behaviour of chicken egg white ovomucoid and its correlation with the three domain structure of the protein

The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and its four fragments representing domains I, II + III, I + II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min-1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.     

Family shuffling of soil DNA to change the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase

Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2'-dichlorobiphenyl (2,2'-CB). In this work, we evolved BPDO by shuffling bphA genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of bphA. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of Burkholderia xenovorans LB400 bphA. Variants were screened for their ability to oxygenate 2,2'-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively cis-5,6-dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl from 2,2'-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2',3,3'-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2',5,5'-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2'-CB at a rate of 16 +/- 1 nmol min(-1) per nmol enzyme, which was similar to the value observed for LB400 BPDO. cis-5,6-Dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues M237 S238 and S283 outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.     

Identification and assignment of base pairs in four helical segments of Bacillus megaterium ribosomal 5S RNA and its ribonuclease T1 cleavage fragments by means of 500-MHz proton homonuclear Overhauser enhancements.

Three different fragments of Bacillus megaterium ribosomal 5S RNA have been produced by enzymatic cleavage with ribonuclease T1. Fragment A consists of helices II and III, fragment B contains helix IV, and fragment C contains helix I of the universal 5S rRNA secondary structure. All (eight) imino proton resonances in the downfield region (9-15 ppm) of the 500-MHz proton FT NMR spectrum of fragment B have been identified and assigned as G80.C92-G81.C91-G82.C90-A83.++ +U89-C84.G88 and three unpaired U's (U85, U86, and U87) in helix IV by proton homonuclear Overhauser enhancement connectivities. The secondary structure in helix IV of the prokaryotic loop is completely demonstrated spectroscopically for the first time in any native or enzyme-cleaved 5S rRNA. In addition, G21.C58-A20.U59-G19.C60-A18.U61 in helix II, U32.A46-G31.C47-C30.G48-C29.G49 in helix III, and G4.C112-G5.C111-U6.G110 in the terminal stem (helix I) have been assigned by means of NOE experiments on intact 5S rRNA and its fragments A and C. Base pairs in helices I-IV of the universal secondary structure of B. megaterium 5S RNA are described.     

Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.     

Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites

Cyclic AMP-dependent protein kinase catalyzes the incorporation of 3-4 mol of phosphate into the alpha subunit of rat brain sodium channels in vitro or in situ. Digestion of phosphorylated sodium channels with CNBr yielded three major phosphorylated fragments of 25, 31, and 33 kDa. These fragments were specifically immunoprecipitated with site-directed antisera establishing their location within an intracellular loop between the first and second homologous domains containing residues 448 to 630 of sodium channel RI or residues 450-639 of sodium channel RII. Five of the seven major tryptic phosphopeptides generated from intact sodium channel alpha subunits were contained in each of the 25-, 31-, and 33-kDa CNBr fragments, indicating that most cAMP-dependent phosphorylation sites are in this domain. Since CNBr digestion of sodium channels which had been metabolically labeled with 32P in intact neurons yielded the same phosphorylated fragments, the phosphorylated region we have identified is the major location of phosphorylation in situ. Only serine residues were phosphorylated by cAMP-dependent protein kinase in vitro, while approximately 16% of the phosphorylation in intact neurons was on threonine residues that must lie outside the domain we have identified. Since this domain is phosphorylated in intact neurons, our results show that it is located on the intracellular side of the plasma membrane. These results are considered with respect to models for the transmembrane orientation of the alpha subunit.     

Interactions of the C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule

The highly selective sodium channel blocker, tetrodotoxin (TTX) has been instrumental in characterization of voltage-gated sodium channels. TTX occludes the ion-permeation pathway at the outer vestibule of the channel. In addition to a critical guanidinium group, TTX possesses six hydroxyl groups, which appear to be important for toxin block. The nature of their interactions with the outer vestibule remains debatable, however. The C-11 hydroxyl (C-11 OH) has been proposed to interact with the channel through a hydrogen bond to a carboxyl group, possibly from domain IV. On the other hand, previous experiments suggest that TTX interacts most strongly with pore loops of domains I and II. Energetic localization of the C-11 OH was undertaken by thermodynamic mutant cycle analysis assessing the dependence of the effects of mutations of the adult rat skeletal muscle Na(+) channel (rNa(v)1.4) and the presence of C-11 OH on toxin IC(50). Xenopus oocytes were injected with the mutant or native Na(+) channel mRNA, and currents were measured by two-electrode voltage clamp. Toxin blocking efficacy was determined by recording the reduction in current upon toxin exposure. Mutant cycle analysis revealed that the maximum interaction of the C-11 OH was with domain IV residue D1532 (DeltaDeltaG: 1.0 kcal/mol). Furthermore, C-11 OH had significantly less interaction with several domain I, II, and III residues. The pattern of interactions suggested that C-11 was closest to domain IV, probably involved in a hydrogen bond with the domain IV carboxyl group. Incorporating this data, a new molecular model of TTX binding is proposed.     

Determination of the distribution of D-glucuronic acid units within the chain of pig-skin dermatan sulfate near the linkage region

A method for analyzing the distribution of D-glucuronic acid units within the chain and near the linkage region of dermatan sulfate has been developed. The method consists of a chemical modification of the reducing terminal residue in the polysaccharide by reductive amination with excess 1,2-diaminoethane in the presence of sodium cyanoborohydride, desulfative fragmentation of the polysaccharide, labeled with 2-aminoethylamino (AEA) groups, in hot dimethyl sulfoxide containing 10% of water followed by 2,4-dinitrophenylation of the 2-aminoethylamino group, separation of the 2-(2,4-dinitrophenylamino)ethylamino labeled dermatan fragments from nonlabeled fragments on Octyl-Sepharose CL-4B gel, and determination of the uronic acid composition of the labeled fragments having various chain-length. A preparation of pig-skin dermatan sulfate (Mr 21,000, ratio of GlcA to total uronic acid, 93:500) showed an average distribution pattern of D-glucuronic acid residues near the linkage region of one N-acetylchondrosine unit in the disaccharide sequence 1-5(6) linked to the Xyl----Gal----Gal----GlcA residue, a cluster of 6-8 N-acetyldermosine units in the sequence 6(7)-12(13), and four separate N-acetylchondrosine units between the sequence adjacent to the N-acetyldermosine cluster and the sequence 23 or higher.     

Mapping the path of the nascent peptide chain through the 23S RNA in the 50S ribosomal subunit.

Peptides of different lengths encoded by suitable mRNA fragments were biosynthesized in situ on Escherichia coli ribosomes. The peptides carried a diazirine derivative bound to their N-terminal methionine residue, which was photoactivated whilst the peptides were still attached to the ribosome. Subsequently, the sites of photo-cross-linking to 23S RNA were analyzed by our standard procedures. The N-termini of peptides of increasing length became progressively cross-linked to nucleotide 750 (peptides of 6, 9 or 13-15 amino acids), to nucleotide 1614 and concomitantly to a second site between nucleotides 1305 and 1350 (a peptide of 25-26 amino acids), and to nucleotide 91 (a peptide of 29-33 amino acids). Previously we had shown that peptides of 1 or 2 amino acids were cross-linked to nucleotides 2062, 2506 and 2585 within the peptidyl transferase ring, whereas tri-and tetrapeptides were additionally cross-linked to nucleotides 2609 and 1781. Taken together, the data demonstrate that the path of the nascent peptide chain moves from the peptidyl transferase ring in domain V of the 23S RNA to domain IV, then to domain II, then to domain III, and finally to domain I. These cross-linking results are correlated with other types of topographical data relating to the 50S subunit.     

Structural investigation of helices II, III, and IV of B. megaterium 5S ribosomal RNA by molecular dynamics calculations.

The structures of the helices II-III region and the helix IV region of B. megaterium 5S rRNA have been examined by means of energy minimization and molecular dynamics calculations. Calculated distances between neighboring hydrogen-bonded imino protons in helices II, III, and IV were between 3.5 and 4.5 A. The overall axis for the helices II-III region is warped rather than straight. Formation of additional Watson-Crick base pairs in loop B and loop C was not evident from the atomic positions calculated by molecular dynamics. Bases in loop C are well stacked, showing no significant change during dynamics. Bulge migration in helix III does not seem to be possible; the helices II-III region prefers one conformation. Helix II is more stable than helix III. Five base pairs in helix IV were sufficiently stable to establish that helix IV is terminated by a hairpin loop of three nucleotides. U87 protrudes from loop D. Structures of the helices II-III segment and the helix IV segment of B. megaterium 5S rRNA obtained by molecular dynamics were generally consistent with the solution structure inferred from high-field proton nmr spectroscopy.     

Molecular action of lidocaine on the voltage sensors of sodium channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Q(max)) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel-gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Q(max) of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near -100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.     

Minimal sodium channel pore consisting of S5-P-S6 segments preserves intracellular pharmacology

We studied the properties of a sodium channel comprised only of S5-P-S6 region of the rat sodium channel alpha-subunit Nav1.4 (micro1pore). Results obtained in HEK cell lines permanently transfected with the sodium channel alpha-subunit or with the micro1pore were compared with data of the native HEK cells. Sodium channel blockers, tetrodotoxin and tetracaine, protect cells transfected with the complete sodium channel against death produced by incubation with veratridine. Veratridine-induced cell death in cell lines expressing the micro1pore construct is antagonised by tetracaine, but not by tetrodotoxin. Whole-cell conductance also increases in the presence of veratridine in micro1pore transfected cells and tetracaine inhibits these currents. Our pharmacological and electrophysiological data suggest that micro1pore keeps binding sites for veratridine and tetracaine, but not for TTX, and reconstitutes the permeation pathway for Na+ ions.     

Cys(577) is a conformationally mobile residue in the ATP-binding domain of the Na,K-ATPase alpha-subunit.

2-[4'-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K(+) (<1 mM); the protective effect K(+) is reversed at higher concentrations. This biphasic effect was also observed with K(+) congeners. In contrast, Na(+) ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of approximately 5 kDa. N-terminal amino acid sequencing identified the peptide (V(545)LGFCH...). This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.     

Organization of rRNA structural genes in the archaebacterium Thermoplasma acidophilum.

In the archaebacterium Thermoplasma acidophilum, each of the structural genes for 5S, 16S and 23S rRNA occur once per genome. In contrast to those of eubacteria and eukaryotes, they appear unlinked. The distance between the 16S and the 23S rDNA is at least 7.5 Kb, that between 23S and 5S rDNA at least 6 Kb and that between 16S and 5S rDNA at least 1.5 Kb. No linkage between those genes has been found by the analysis of recombinant plasmids carrying Bam HI and Hind III rDNA fragments as by hybridizing those plasmids to fragments of Thermoplasma DNA generated by 6 individual restriction endonucleases, recognizing hexanucleotide sequences.     

Ribonucleoprotein organization of eukaryotic RNA. XXXI. Structure of the U1 small nuclear ribonucleoprotein

A small nuclear ribonucleoprotein, U1 snRNP, has been implicated in mRNA processing. In this investigation sites of protein binding on U1 RNA were mapped by nuclease protection and RNA sequencing. Partially purified human U1 snRNP was sequentially digested with Escherichia coli RNAase III and S1 nuclease. The resistant ribonucleoprotein fragments were deproteinized, preparatively hybridized to the U1 RNA--complementary DNA strand of a human U1 gene cloned in bacteriophage M13, and displayed by electrophoresis. The nuclease-resistant U1 RNA fragments were between 23 and 63 nucleotides in length. Most of these fragments were not obtained when protein-free U1 RNA was similarly digested, whereas others were obtained in low yield from U1 RNA and much higher yield from U1 snRNP. RNA sequencing of the fragments revealed that the protein-protected sites in U1 snRNP correspond to base-paired stems I and II, loop a, and portions of stems III and IV (secondary structure nomenclature of Branlant et al., 1981). Single, "bulged" pyrimidines are present within the protein-covered helical regions of stems I and III. Most interestingly, the single-stranded 5' end of U1 RNA, implicated in mRNA splicing, was also highly protected by protein. These results demonstrate that the great majority of U1 RNA is covered by protein in U1 snRNP. The association of protein with the 5' end of U1 RNA is in agreement with recent evidence that snRNP proteins potentiate the binding of this region of U1 RNA with pre-mRNA splice sites.