Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.     

Three-dimensional structure of Gln25-ribonuclease T1 at 1.84-A resolution: structural variations at the base recognition and catalytic sites.

The structure of the Gln25 variant of ribonuclease T1 (RNase T1) crystallized at pH 7 and at high ionic strength has been solved by molecular replacement using the coordinates of the Lys25-RNase T1/2'-guanylic acid (2'GMP) complex at pH 5 [Arni et al. (1988) J. Biol. Chem. 263, 15358-15368] and refined by energy minimization and stereochemically restrained least-squares minimization to a crystallographic R-factor of 14.4% at 1.84-A resolution. The asymmetric unit contains three molecules, and the final model consists of 2302 protein atoms, 3 sulfates (at the catalytic sites), and 179 solvent water molecules. The estimated root mean square (rms) error in the coordinates is 0.15 A, and the rms deviation from ideality is 0.018 A for bond lengths and 1.8 degrees for bond angles. Significant differences are observed between the three molecules in the asymmetric unit at the base recognition and catalytic sites.     

Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.     

Computer modeling studies of ribonuclease T1-guanosine monophosphate complexes

The three-dimensional structures of ribonuclease (RNase) T1 complexes with the inhibitors 2'-guanylic acid (2'-GMP), 3'-guanylic acid (3'-GMP), and 5'-guanylic acid (5'-GMP) were predicted by energy minimization studies. It is shown that these inhibitors can bind to RNase T1 in either of the ribose puckered conformations (C2'-endo and C3'-endo) in solid state and exist in significant amounts in both forms in solution. These studies are in agreement with the x-ray crystallographic studies of the 2'-GMP-Lys25-RNase T1 complex, where the inhibitor binds in C2'-endo puckered conformation. These results are also in good agreement with the available 1H-nmr results of Inagaki et al. [(1985) Biochemistry 24, 1013-1020], but differ from their conclusions where the authors favor only the C3'-endo ribose conformation for all the three inhibitors. The calculations explain the apparent discrepancies in the conclusions drawn by x-ray crystallographic and spectroscopic studies. An extensive hydrogen-bonding scheme was predicted in all the three complexes. The hydrogen-bonding scheme predicted for the 2'-GMP (C2'-endo)-RNase T1 complex agrees well with those reported from x-ray crystallographic studies. In all three complexes the base and the phosphate bind in nearly identical sites independent of the position of the phosphate or the ribose pucker. The glycosyl torsion angle favors a value in the +syn range in the 2'-GMP (C2'-endo)-RNase T1, 3'-GMP (C2'-endo)-RNase T1, and 3'-GMP (C3'-endo)-RNase T1 complexes; in the high-syn range in the 2'-GMP (C3'-endo)-RNase T1 complex; and in the -syn range in the 5'-GMP (C2'-endo)-RNase T1 and 5'-GMP (C3'-endo)-RNase T1 complexes. These results are in agreement with experimental studies showing that the inhibitory power decreases in the order 2'-GMP greater than 3'-GMP greater than 5'-GMP, and they also explain the high pKa value observed for Glu58 in the 2'-GMP-RNase T1 complex.     

Conformation of 2'GMP bound to a mutant ribonuclease T1 (Y45W) determined by X-ray diffraction and NMR methods.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 A resolution to a conventional R-factor of 0.164. The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP. The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base. The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP. Analysis of 500-MHZ NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution [Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020].     

Quantitative analysis of the contribution of Glu46 and Asn98 to the guanosine specificity of ribonuclease T1

In the crystal structure of the ribonuclease T1 (RNase T1; EC 3.1.27.3)-2'-GMP complex the hydrogen-bonding potential of the guanine base is saturated [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368]. The oxygens of the Glu46 carboxylate and the Asn98 main-chain carbonyl act as hydrogen-bond acceptors for the N(1)H-C(2)-N(2)H2 part of the base. We measured the transesterification kinetics of wild-type and Glu46Ala RNase T1 using the GpU, IpU, and XpU series of analogous substrates. We found that the N(1)H---Glu46 O epsilon 1, the N(2)H---Glu46 O epsilon 2, and the N(2)H---Asn98 O hydrogen bonds have an apparent contribution of 2.7, 1.1, and 1.2 kcal/mol to the interaction energy of the enzyme and the transition state of the substrate. Wild-type RNase T1 discriminates guanine from nonionized xanthine (a guanine analogue in which the exocyclic amino group is replaced by an oxygen) by about 4.4 kcal/mol. Loss of the specific hydrogen bonds with the exocyclic amino group of the guanine base accounts for 2.4 kcal/mol of this discrimination energy; 2.0 kcal/mol is due to unfavorable non-H-bonded oxygen-oxygen contacts in the enzyme-xanthine complex. A pH dependence study shows that the deprotonated form of xanthine (i.e., the 6-keto-2-enolate anion; pKa = 5.4) is far less preferred, if not excluded, as substrate by wild-type RNase T1; this may be attributed to an electrostatic repulsion of the negatively charged xanthine by the Glu46 carboxylate group.     

Computer modelling studies on the mechanism of action of ribonuclease T1.

The mechanism of action of ribonuclease (RNase) T1 is still a matter of considerable debate as the results of x-ray, 2-D nmr and site-directed mutagenesis studies disagree regarding the role of the catalytically important residues. Hence computer modelling studies were carried out by energy minimisation of the complexes of RNase T1 and some of its mutants (His40Ala, His40Lys, and Glu58Ala) with the substrate guanyl cytosine (GpC), and of native RNase T1 with the reaction intermediate guanosine 2',3'-cyclic phosphate (G greater than p). The puckering of the guanosine ribose moiety in the minimum energy conformer of the RNase T1-GpC (substrate) complex was found to be O4'-endo and not C3'-endo as in the RNase T1-3'-guanylic acid (inhibitor/product) complex. A possible scheme for the mechanism of action of RNase T1 has been proposed on the basis of the arrangement of the catalytically important amino acid residues His40, Glu58, Arg77, and His92 around the guanosine ribose and the phosphate moiety in the RNase T1-GpC and RNase T1-G greater than p complexes. In this scheme, Glu58 serves as the general base group and His92 as the general acid group in the transphosphorylation step. His40 may be essential for stabilising the negatively charged phosphate moiety in the enzyme-transition state complex.     

DNA adduct bypass polymerization by Sulfolobus solfataricus DNA polymerase Dpo4: analysis and crystal structures of multiple base pair substitution and frameshift products with the adduct 1,N2-ethenoguanine

1,N(2)-Etheno(epsilon)guanine is a mutagenic DNA lesion derived from lipid oxidation products and also from some chemical carcinogens. Gel electrophoretic analysis of the products of primer extension by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) indicated preferential incorporation of A opposite 3'-(1,N(2)-epsilon-G)TACT-5', among the four dNTPs tested individually. With the template 3'-(1,N(2)-epsilon-G)CACT-5', both G and A were incorporated. When primer extension was done in the presence of a mixture of all four dNTPs, high pressure liquid chromatography-mass spectrometry analysis of the products indicated that (opposite 3'-(1,N(2)-epsilon-G)CACT-5') the major product was 5'-GTGA-3' and the minor product was 5'-AGTGA-3'. With the template 3'-(1,N(2)-epsilon-G)TACT-5', the following four products were identified by high pressure liquid chromatography-mass spectrometry: 5'-AATGA-3', 5'-ATTGA-3', 5'-ATGA-3', and 5'-TGA-3'. An x-ray crystal structure of Dpo4 was solved (2.1 A) with a primer-template and A placed in the primer to be opposite the 1,N(2)-epsilon-G in the template 3'-(1,N(2)-epsilon-G)TACT 5'. The added A in the primer was paired across the template T with classic Watson-Crick geometry. Similar structures were observed in a ternary Dpo4-DNA-dATP complex and a ternary Dpo4-DNA-ddATP complex, with d(d)ATP opposite the template T. A similar structure was observed with a ddGTP adjacent to the primer and opposite the C next to 1,N(2)-epsilon-G in 3'-(1,N(2)-epsilon-G)CACT-5'. We concluded that Dpo4 uses several mechanisms, including A incorporation opposite 1,N(2)-epsilon-G and also a variation of dNTP-stabilized misalignment, to generate both base pair and frameshift mutations.     

Three-dimensional structure of ribonuclease T1 complexed with an isosteric phosphonate substrate analogue of GpU: alternate substrate binding modes and catalysis

The X-ray crystal structure of a complex between ribonuclease T1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2. 0 A resolution. This ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. On the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(Sp)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [Steyaert, J., and Engleborghs (1995) Eur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.     

Crystal structure disposition of thymidylic acid tetramer in complex with ribonuclease A.

The crystal structure of ribonuclease A with bound thymidylic acid tetramer is reported at 2.5-A resolution. The diffusion of the tetramer into native orthorhombic crystals of the ribonuclease allows for the formation of a structurally stable complex where the single-stranded nucleic acid enters and leaves the enzyme's catalytic region in a persistent 5'-3' direction. The binding of the tetramer to the enzyme's surface is facilitated and mediated by electrostatic interactions between basic protein residues and nucleotide phosphates. Two pyrimidine nucleotides are bound to the enzyme's active site in a manner similar to that observed for other complexes between ribonuclease A and nucleic acid oligomers.     

NMR study of the positions of His-12 and His-119 in the ribonuclease A-uridine vanadate complex.

The binding of uridine vanadate to ribonuclease A has been investigated by one- and two-dimensional 1H NMR. The homonuclear Nuclear Overhauser and exchange spectroscopy spectrum of the uridine vanadate/RNase A complex exhibits cross peaks between both the C5H and C6H protons of uridine vanadate and the H epsilon 1 proton of His-12 of ribonuclease A. These cross peaks suggest that the H epsilon 1 proton of His-12 is in the vicinity of the uracil base of uridine vanadate, as observed in the crystallographic structure of the uridine vanadate/RNase A complex. However, no cross peaks are observed between the C5H and C6H protons of uridine vanadate and the H epsilon 1 proton of His-119 of ribonuclease A, although they were predicted based upon the distances calculated from coordinates of the crystallographic structure of the complex. These results suggest that there is a significant difference between the positioning of the His-119 side chain in the solution and in the crystallographic structures.     

The specific guanine binding site of the ribonuclease T1 family enzymes and of G-proteins is modeled in the cocrystal formed by 7-methylguanosine-5'-phosphate and phenylalanine

In the cocrystal formed by 7-methylguanosine-5'-phosphate.phenylalanine.6H2O, the interactions between guanine and phenylalanine are similar to those observed in the complex of ribonuclease T1 with 2'-guanylic acids, and those of the two G-proteins, Elongation Factor-Tu and ras oncogene p21, with GDP. They are similar in the following three points: (a) guanine N(1)H and N(2)H donate cyclic N-H...O hydrogen bonds to the carboxylate group of phenylalanine in the former cocrystal and to the side chain carboxylate group of Asp or Glu in the latter proteins, (b) O(6) of guanine accepts hydrogen bond(s) from main-chain NH group(s), and (c) the purine moiety is sandwiched between aromatic (or hydrophobic) amino acid side chains.     

Crystallographic characterization of an exocyclic DNA adduct: 3,N4-etheno-2'-deoxycytidine in the dodecamer 5'-CGCGAATTepsilonCGCG-3'

Exocyclic DNA adducts are formed from metabolites of chemical carcinogens and have also been detected as endogenous lesions in human DNA. The exocyclic adduct 3,N(4)-etheno-2'-deoxycytidine (epsilon dC), positioned opposite deoxyguanosine in the B-form duplex of the dodecanucleotide d(CGCGAATTepsilonCGCG), has been crystallographically characterized at 1.8A resolution. This self-complementary oligomer crystallizes in space group P3(2)12, containing a single strand in the asymmetric unit. The crystal structure was solved by isomorphous replacement with the corresponding unmodified dodecamer structure. Exposure of both structures to identical crystal packing forces allows a detailed investigation of the influence of the exocyclic base adduct on the overall helical structure and local geometry. Structural changes are limited to the epsilon C:G and adjacent T:A and G:C base-pairs. The standard Watson-Crick base-pairing scheme, retained in the T:A and G:C base-pairs, is blocked by the etheno bridge in the epsilon C:G pair. In its place, a hydrogen bond involving O2 of epsilon C and N1 of G is present. Comparison with an epsilon dC-containing NMR structure confirms the general conformation reported for epsilon C:G, including the hydrogen bonding features. Superposition with the crystal structure of a DNA duplex containing a T:G wobble pair shows similar structural changes imposed by both mismatches. Evaluation of the hydration shell of the duplex with bond valence calculations reveals two sodium ions in the crystal.     

Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study.

The tryptophyl fluorescence of ribonuclease T1 decays monoexponentially at pH 5.5, tau = 4.04 ns but on increasing pH, a second short-lived component of 1.5 ns appears with a midpoint between pH 6.5 and 7.0. Both components have the same fluorescence spectrum. Acrylamide quenches both fluorescence components, and the short-lived component is quenched fivefold faster than the predominant long component. Binding of the substrate analogue 2'-guanylic acid at pH 5.5 quenches the fluorescence by 20% and introduces a second decay component, tau = 1.16 ns. Acrylamide quenches both tryptophyl decay components, with similar quenching rates. The fluorescence anisotropy decay of ribonuclease T1 was consistent with a molecule the size of ribonuclease T1 surrounded by a single layer of water at pH 7.4, even though the anisotropy decay at pH 5.5 deviated from Stokes-Einstein behavior. The fluorescence data were interpreted with a model where the tryptophyl residue exists in two conformations, remaining in a hydrophobic pocket. The acrylamide quenching is interpreted with electron transfer theory and suggests that one conformer has the nearest atom approximately 3 A from the protein surface, and the other, approximately 2 A.     

A comparison of the different DNA binding specificities of the bZip proteins C/EBP and GCN4.

The bZip proteins GCN4 and C/EBP differ in their DNA binding specificities: GCN4 binds well to the pseudopalindromic AP1 site 5'-A4T3G2A1C0T1C2'A3'T4'-3' and to the palindromic ATF/CREB sequence 5'-A4T3G2A1-C0*G0'T1'C2'A3'T4'-3'; C/EBP preferentially recognizes the palindromic sequence 5'-A4T3T2G1C0*G0'C1'A2'-A3'T4'-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in specific base contacts: asparagine -18, alanine -15, alanine -14, serine -11 and arginine -10 (numbered relative to the start point of the leucine zipper, which we define as +1). In the basic region of C/EBP position -14 is occupied by valine instead of alanine, the other four residues being identical. Here we analyse the role of valine -14 in C/EBP-DNA complex formation. Starting from a C/EBP-GCN4 chimeric bZip peptide which displays C/EBP specificity, we systematically mutated position -14 of its basic region and characterized the DNA binding specificities of the 20 possible different peptides by gel mobility shift assays with various target sites. We present evidence that valine -14 of C/EBP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4, which exclusively contacts thymine 1' of the GCN4 binding sites.     

Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus.

The crystal structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.5 A resolution by the multiple isomorphous replacement method. The crystal structure was refined by simulated annealing with molecular dynamics. The current crystallographic R-factor is 0.200 in the 10-2.5 A resolution range. The molecular structure which is completely different from the known structures of RNase A and RNase T1 consists of six alpha-helices and seven beta-strands, belonging to the alpha+beta type structure. Two histidine and one glutamic acid residues which were predicted as the most probably functional residues by chemical modification studies are found to be clustered. The steric nature of the active site taken together with the relevant site-directed mutagenesis experiments (Irie et al.) indicates that: (i) the two histidine residues are the general acid and base; and (ii) an aspartic acid residue plays a role of recognizing adenine moiety of the substrate.     

Biochemical and genetic studies on GP43, a 43-kD glycoprotein detected immunologically in human urine and serum

A new and previously undescribed glycoprotein with a molecular weight of 43,000 has been isolated from human urine. This protein, designated GP43; copurified with ribonuclease, which has the same molecular weight, but ribonuclease activity was removed by passage through an affinity column of agarose-5'-(4-aminophenyl phosphoryl) uridine 2'(3') phosphate. GP43 contains about 5.9% neutral sugar, 2.3% hexosamine, and 1.6% sialic acid. A rabbit antibody to the purified GP43 reacted with human urine and serum as well as with the purified GP43. The genetic polymorphism of GP43 was then studied in desialylated human serum samples by urea-polyacrylamide gel isoelectric focusing, followed by immunoblotting with the specific antibody for GP43. Three common phenotypes, designated GP43 1, 1-2, and 2, were easily recognized using this technique and represented homozygosity or heterozygosity for two autosomal codominant alleles, GP43*1 and GP43/2. The frequencies of the GP43*1 and GP43*2 alleles in a Japanese population were 0.7683 and 0.2317, respectively.     

Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex with 2'GMP.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific complex of the enzyme with 2'GMP was also determined and refined to an R-factor of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a base-binding site that is far apart from the guanine recognition site, where the guanine base of 2'GMP binds. The binding of the adenine base is mediated by a single hydrogen bond and stacking interaction of the base with the imidazole ring of His92. The mode of stacking of the adenine base with His92 is similar to the stacking of the guanine base observed in complexes of ribonuclease T1 with guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases, reported by Lenz et al., and in the complex of barnase with d(GpC), reported by Baudet & Janin. These observations suggest that the site is non-specific for base binding. The phosphate group of 2'AMP is tightly locked at the catalytic site with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with conformational changes of the sugar ring and displacement of the phosphate group towards the interior of the catalytic site from the corresponding position in the wild-type enzyme complex. Comparison of two crystal structures obtained provides a solution to the problem that non-cognizable nucleotides exhibit unexpectedly strong binding to the enzyme, compared with high specificity in nucleolytic activity. The results indicate that the discrimination of the guanine base from the other nucleotide bases at the guanine recognition site is more effective than that estimated from nucleotide-binding experiments so far.     

5S RNA structure and interaction with transcription factor A. 1. Ribonuclease probe of the structure of 5S RNA from Xenopus laevis oocytes

The structure of Xenopus laevis oocyte (Xlo) 5S ribosomal RNA has been probed with single-strand-specific ribonucleases T1, T2, and A with double-strand-specific ribonuclease V1 from cobra venom. The digestion of 5'- or 3'-labeled renatured 5S RNA samples followed by gel purification of the digested samples allowed the determination of primary cleavage sites. Results of these ribonuclease digestions provide support for the generalized 5S RNA secondary structural model derived from comparative sequence analysis. However, three putative single-stranded regions of the molecule exhibited unexpected V1 cuts, found at C36, U73, U76, and U102. These V1 cuts reflect additional secondary structural features of the RNA including A.G base pairs and support the extended base pairing in the stem containing helices IV and V which was proposed by Stahl et al. [Stahl, D. A., Luehrsen, K. R., Woese, C. R., & Pace, N. R. (1981) Nucleic Acids Res. 9, 6129-6137]. A conserved structure for helix V having a common unpaired uracil residue at Xlo position 84 is proposed for all eukaryotic 5S RNAs. Our results are compared with nuclease probes of other 5S RNAs.     

Secondary structure model for mouse beta Maj globin mRNA derived from enzymatic digestion data, comparative sequence and computer analysis.

A model for the secondary structure of mouse beta Maj globin messenger RNA is presented based on enzymatic digestion data, comparative sequence and computer analysis. Using 5'-32P-end-labeled beta globin mRNA as a substrate, single-stranded regions were determined with S1 and T1 nucleases and double-stranded regions with V1 ribonuclease from cobra venom. The structure data obtained for ca. 75% of the molecule was introduced into a computer algorithm which predicts secondary structures of minimum free energy consistent with the enzymatic data. Two prominent base paired regions independently derived by phylogenetic analysis were also present in the computer generated structure lending support for the model. An interesting feature of the model is the presence of long-range base pairing interactions which permit the beta globin mRNA to fold back on itself, thereby bringing the 5'- and 3'-noncoding regions within close proximity. This feature is consistent with data from other laboratories suggesting an interaction of the 5'- and 3'-domains in the mammalian globin mRNAs.