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At least 19 recordsLinked to original sources
PubMed1979

Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1.

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41-45 (1979) and J. Biol. Chem. 254, 4309-4312 (1979). The mutant (m1-S1) contains 77% and the fragment (S1-F1) 66% of the polypeptide chain length (approximately 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that m1-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and phi X174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or phi X174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.

Bacterial Proteins↗
PubMed1977

Nucleic acid helix-unwinding properties of ribosomal protein S1 and the role of S1 in mRNA binding to ribosomes.

The presence of ribosomal protein S1 in 30S ribosomes is indispensable for the formation of 30S initiation complexes with natural mRNA. The 30S subunits lacking S1 retain activity with AUG as mRNA and are also active in poly(rU)-directed binding of Phe-tRNA. Isolated protein S1 stoichiometrically disrupts the secondary structure of helical and stacked single-stranded polynucleotides and converts them into their fully or partially denatured forms. A mono-N-ethylmaleimide derivatives of S1 is nearly devoid of any RNA helix-unwinding properties but is readily incorporated into 30S subunits deficient in S1. The resulting N-ethylmaleimide-S1-containing 30S subunits are completely inactive in the binding of MS2 [3H]RNA and in the formation of an initiation complex with MS2 RNA as mRNA. They retain activity in the binding of the initiator fMet-tRNA in response to the trinucleotide AUG and in the binding of Phe-tRNA in response to poly(U). They also retain the capacity to bind 50S subunits and to form 70S couples. These results suggest that a correlation exists between the RNA helix-unwinding capacity of isolated S1 and the function of S1 in the ribosomal binding of natural mRNA when the protein becomes part of the 30S subunit.

Binding Sites↗
PubMed1977

[Substrate inhibition as a cause of oscillations in an open irreversible enzymic reaction S1 + S2 in the presence of E(R,T) leads to S1' + S2'. A mathematical model].

A mathematical model of an open irreversible reaction S1 + S2 (formula: see text) catalysed by an olygomeric enzyme E(R, T) has been analysed. It is assumed that the enzyme undergoes the concerted conformational transitions R in equilibrium T in conformity with the theory of Monod, Wyman and Changeux, and one of the substrates (S2) produces inhibition of the enzyme, thus shifting the equilibrium between the two enzyme forms in the direction of T formation. A simple graphical explanation is given to the hysteresis of the input characteristic approximately v ([S1]) (approximately v is the reaction rate at d[S2]/dt=O) which gives rise to self--oscillations. The hysteresis occurs both in the case of allosteric and isosteric substrate inhibition.

Allosteric Regulation↗
PubMed1979

Segmental flexibility in Escherichia coli ribosomal protein S1 as studied by fluorescence polarization.

Ribosomal protein S1 covalently reacts with approximately one equivalent of iodoacetylethylenediamine (1,5-napthol sulfonate (IAEDANS) or iodoacetylaminofluorescein (IAAF). The product AEDANS-S1 can bind to 30S ribosomal subunits lacking S1 as shown by polyacrylamide-agarose gel electrophoresis AEDANS-S1 and AAF-S1 when added back to S1-depleted 30S subunits modulate poly(U)-dependent polyphenylalanine synthesis in the presence of IF3 in a very similar way to unmodified S1. AEDANS-S1 also stimulates RI7-dependent fMet-tRNA binding to 1.0M NH4C1 washed ribosomes whereas AAF-S1 does not. Both static and nanosecond fluorescence polarization techniques were used to study the rotational motions of AEDANS-S1. Several previous studies had indicated that S1 is a highly extended protein which can be modeled by a prolate ellipsoid with an axial ratio of 10 to 1. However, the rotational correlation time we find is about half that expected for such a particle. This suggests that S1 is a flexible protein with at least two domains that can rotate independently.

Escherichia coli↗
PubMed1979

Interaction of Escherichia coli ribosomal protein S1 with ribosomes.

The binding affinity of Escherichia coli ribosomal protein S1 for 30S ribosomal particles has been determined by a sucrose gradient band sedimentation technique; the association constant (K) for the binding of one S1 protein per active 30S ribosomal subunit is approximately 2 X 10(8) M-1. The involvement of the two polynucleotide binding sites of S1 protein (site I binding single-stranded DNA or RNA, and site II binding single-stranded RNA only) in the S1--ribosomal interaction have been examined by competition experiments with polynucleotides of known affinity for the two sites. We find that site I does not contribute to the interaction; site II binding appears to provide a major part of the binding free energy, presumably by interaction of S1 with the 16S rRNA of the 30S particle. The remaining binding free energy is probably derived from the interaction of S1 protein with other proteins of the 30S subunit. The affinity of S1 for 70S ribosomes is about the same as that for the 30S subunit; the affinity of S1 for 50S subunits is much less. Binding affinities and stoichiometries of S1 protein with "inactive" 30S ribosomal subunits have also been examined.

Binding, Competitive↗
PubMed1976

Thiols of myosin. IV. "Abnormal" reactivity of S1 thiol and the conformational changes around S2 thiol.

The flexibility of the tertiary structure around the active site of myosin ATPase [EC 3.6.1.3] was studied using the reactivity of two specific thiol groups, S1 and S2, as a structural probe. The following four maleimide derivatives were used as thiol-directed reagents: N-ethylmaleimide (NEM), N-(4-methoxy-2-benzimidazolyl methyl) maleimide (MBM), N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM) and N-(4-dimethyl-amino-3,5-dinitrophenyl)maleimide (DDPM). 1. All the maleimide derivatives used activated the Ca2+-ATPase activity and inhibited the EDTA-ATPase activity, like NEM, indicating that they modified S1. The rate of modification of S1 by NEM and BIPM increased with increasing pH, while that by DDPM decreased. BIPM simultaneously modified S1 and S2. 2. S1 showed much higher reactivity toward the maleimides, except for BIPM, than did N-acetylcysteine (N-Ac-Cys) a low molecular-weight model compound. The extremely small pKa value of S1, 6.28, accounted for this high reactivity. In addition, the ATP-induced increase in its reactivity inducated that S1 was in a buried state. Kinetic analysis showed that the teritiary structure around S1 at alkaline pH differed from that at acidic pH. 3. The apparent rate constant of S2-modification with NEM was approximately one seven-hundredth and one four-hundredth of those of S1 and N-Ac-Cys, respectively. Fluorimetric studies using BIPM revealed that S2 in the buried state was exposed upon adding ATP; this was compensated by the burying of some other thiol group(s) (Sp). Non-linearity of the Arrhenius plots of the reaction rate of S2 suggested that the S2 region of myosin had different conformations at high and low temperatures, the transition temperature being 10--15degrees. This non-linearity completely disappeared in the presence of Mg2+-ATP. On the other hand, Arrhenius plots for the thiols reactive to BIPM did not show non-linearity in the presence or absence of ATP.

Acetylcysteine↗
PubMed1979

Studies on 30S ribosomal protein S1 from E. coli. I. Purification and physicochemical properties.

1. The distribution of ribosomal protein S1 in subcellular fractions of E. coli was determined by radioimmunoassay. It was found that about 70%, 20% and 10% of protein S1 were present in the high salt (1.0 M NH4Cl)-washed ribosomes, the ribosomal wash and the S100 fraction, respectively. 2. Protein S1 was purified from unwashed ribosomes by an improved procedure which included: (i) extraction of protein S1 from unwashed ribosomes with 1.2 M LiCl and 1.0 M NH4Cl, (ii) ammonium sulfate fractionation, (iii) two successive column chromatographies on DEAE-Sephadex, and (iv) hydroxylapatite column chromatography. Purified protein S1 was homogeneous in polyacrylamide gel electrophoresis under native and denatured conditions. 3. The molecular weights determined by sedimentation equilibrium and by SDS-polyacrylamide gel electrophoresis were 83,000 and 70,000 respectively. The sedimentation coefficient was estimated as 3.0S by glycerol gradient centrifugation. The stokes radius determined by Sephadex G-200 gel filtration was 45 A. From these data, the frictional ratio of protein S1 was calculated to be 1.65, assuming the molecular weight and partial specific volume to be 70,000 and 0.736, respectively. Protein S1 had an elongated shape with an axial ratio of approximately 8.5. 4. Protein S1 contained 2 residues of half-cystine and about 10 residues of tryptophan. From CD measurements, the contents of alpha-helix and beta-structure were estimated to be 32 and 27%, respectively. 5. As reported by Kolb et al. (1977) (Proc. Natl. Acad. Sci. U.S. 74, 2379-2383), and Draper et al. (1977) (Proc. Natl. Acad. Sci. U.S. 74, 4786-4790), the intrinsic fluorescence of protein S1 was markedly quenched on interaction with poly(U). The maximal quenching was observed when 30 mol of poly(U) (as UMP residues) was added to one mol of the protein.

Amino Acids↗
PubMed1979

DNA from recombinogenic lambda bacteriophages generated by arl mutant of Escherichia coli is cleaved by single-strand-specific endonuclease S1.

When propagated on arl strains (a subclass of Escherichia coli hyper-rec mutants), lambda "Red-" duplication phages accumulated an enhanced potential for recombination. The physical properties of the recombinogenic phages thus obtained ("Arl-" phages) were similar to those of phages grown on arl+ bacteria. However, Arl- phage DNA was cleaved by endonuclease S1 under conditions such that the nuclease is specific for single-stranded DNA;DNA from control phages was S1-resistant. The number of S1 sites (defined by the apparent decrease in single-strand molecular weight) reached a maximum (seven to nine sites per strand of lambda DNA) after five or six rounds of growth on arl bacteria. Similarly, the recombinogenicity of Arl- phages reached a limiting value (recombination frequency, 15%) that was 5 times that of Arl+ phages. Recombinogenicity and S1 susceptibility were accumulated concomitantly during growth on arl+ bacteria. If all increased recombination occurred at the S1 sites, then these regions (about 40 bases each) were about 300 times as recombinogenic as normal DNA regions of the same size, and 1.5 times as recombinogenic as UV-induced lesions. Chromosomal DNA and plasmid DNA (pBR322) from arl cells were more susceptible to nuclease S1 than was DNA from arl+ bacteria. Analysis of the cleavage products suggests that the S1 sites on Arl- lambda phage DNA are located randomly.

Bacteriophage lambda↗
PubMed1977

Involvement of 30S ribosomal protein S1 in poly(U)-directed polyphenylalanine synthesis.

The effect of 30S ribosomal protein S1 on poly(U)-directed polyphenylalanine synthesis was studied using a highly purified cell-free system which was devoid of endogenous S1. The system consisted of homogeneous preparations of EF-Tu, EF-Ts, and EF-G, and 70S ribosomes from which protein S1 had been removed by poly(U)-cellulose column chromatography. It was found that protein S1 was indispensable for translation of poly(U) by an S1-depleted system at low concentrations of poly(U). On the other hand, at higher concentrations of poly(U), a considerable amount of polyphenylalanine was synthesized in the absence of added S1. The stimulatory effect of S1 was observed at all Mg2+ concentrations examined but was most pronounced at 10 mM Mg2+. Some physicochemical properties of the protein were also studied. It was demonstrated that the protein has an elongated shape with an axial ratio of approximately 8.5.

Escherichia coli↗
PubMed1978

Involvement of ribosomal protein S1 in the assembly of the initiation complex.

Antibodies against ribosomal protein S1 (anti-S1) have been used to determine the function of S1 in the partial reactions involved in the translation of MS2 RNA in vitro. Vacant ribosomes are fully sensitive to the antibodies, whereas elongating ribosomes are resistant. We have determined at which stage of translation the resistance to anti-S1 is acquired. We find that insensitivity to anti-S1 already arises upon mixing 30-S subunits with MS2 RNA. Apparently the two particles form a complex in which S1 is functionally protected against its antibody. Complex formation depends on elevated temperature, a suitable ionic environment and it is stimulated by the initiation factor IF-3. It does not depend on IF-1, IF-2 or fMet-tRNA. Thus ribosomes have the potential to recognize the messenger in the absence of fMet-tRNA. Protein S1 appears directly involved in this primary recognition reaction.

Antibodies↗
PubMed1978

Inactivation of the ribosomal protein S1 in polyuridylate binding by reductive methylation of the lysyl-ammonium groups.

The ribosomal protein S1 was modified by reductive methylation of some of its lysyl ammonium groups (S1). With 6 out of 30 groups methylated the protein lost its capacity to form stable complexes with polyuridylate. Addition of excess polyuridylate inhibited the methylation of the lysyl groups. In equilibrium dialysis experiments it was shown that the binding constant between S1 and U15 was lowered 10-fold as compared to the native protein. The pH-dependence of the complex formation between S1 and U15 confirms a participation of the lysyl residues. When S1 depleted 30-S ribosomes were reconstituted with methylated S1 these ribosomes were inactive in the poly(U) stimulated Phe-tRNA binding. The data are discussed with respect to a grid-like interaction between the lysyl groups of the protein and the phosphodiester bonds of the polynucleotide as a molecular basis of protein nucleic acid interaction.

Hydrogen-Ion Concentration↗
PubMed1979

S1 nuclease as a probe of yeast ribosomal 5 S RNA conformation.

5 S RNA was isolated from Saccharomyces cerevisiae grown in the presence of 32P-phosphate and digested with nuclease S1, a single-strand specific nuclease. Two different procedures were employed to determine the sites of attack on the RNA. First, 5 S RNA was isolated from nuclease S1 digests, digested to completion with ribonuclease T1, and then 'fingerprinted' by two-dimensional electrophoresis. Quantitation of each of the characteristic RNAase T1-derived oligonucleotides was employed to determine the relative susceptibility of various regions of the molecule to nuclease S1. A second procedure to define nuclease S1-susceptible sites in the molecule employed polyacrylamide gel electrophoretic fractionation of nuclease S1 digests followed by identification of the nucleotide sequences of the released RNA fragments. Both procedures showed that the region of the molecule between residues 9 and 60 was most susceptible to nuclease S1, with preferential cleavage occurring between residues 12-25 and 50-60. These results are discussed in relation to a proposed model for the secondary structure of yeast 5 S RNA.

Base Sequence↗
PubMed1976

Alteration of polynucleotide secondary structure by ribosomal protein S1.

Ribosomal 30S protein S1 causes disruption of the secondary structure of certain pyrimidine-containing polynucleotides. Helical poly(U), poly(C, U), and neutral and acidic poly(C) are stoichiometrically converted by S1 to structures indistinguishable from their partially or completely thermally denatured forms, as revealed by circular dichroism. Of the several double- and triple-stranded helical polynucleotides tested that contain one polypurine strand and at least one polypyrimidine strand, only the conformation of the DNA.RNA hybrid, poly(A)-poly(dT), is perturbed. In the presence of S1, this hybrid undergoes a transition to a new structure that has a circular dichroism spectrum unlike either the native or thermally denatured forms. Intercalated ethidium bromide is released from poly(A)-poly(dT) by S1, confirming the occurrence of a conformational rearrangement. The translation inhibitor, autintricarboxylic acid, completely inhibits the action of S1 on polypyrimidines, but has no effect on the conformational perturbation of poly(A(-poly(dT). The possible relation between these observations and the biological function of protein S1 is discussed.

Circular Dichroism↗
PubMed1977

Requirement of chain initiation factor 3 and ribosomal protein S1 in translation of synthetic and natural messenger RNA.

Amino acid incorporation directed by poly(A), poly(U) or R17 RNA has been examined in S1-depleted protein synthesizing systems. We observe that the translation of either synthetic or natural messenger RNA is strictly dependent on the presence of chain initiation factor 3 and ribosomal protein S1. With poly(A) or poly(U) both IF-3 and S1 stimulate amino acid incorporation at least 25-fold, and with R17 RNA the stimulation is approximately 15-fold. More than one copy of S1 per ribosome decreases amino acid incorporation directed by poly(U) or R17 RNA. Initiation complex formation with R17 RNA is also stimulated optimally by the addition of one copy of S1 per ribosome. The function of IF-3 and S1 in protein synthesis is considered.

Bacterial Proteins↗
PubMed1979

The 3' terminus of 16S rRNA: secondary structure and interaction with ribosomal protein S1.

We report studies of the secondary structure and S1 ribosomal protein binding properties of the colicin fragment, containing 49 residues from the 3' terminus of E. coli 16S rRNA. Temperature jump relaxation kinetic measurements reveal two helices in the structure. One of these, melting at 81 degrees C in 5 mM Mg2+, is associated with the 9-base pair hairpin helix predicted by the nucleotide sequence. The other melting transition, at 21 degrees C in 5 mM Mg2+, is assigned to a 4-base pair helix which constrains the pyrimidine tract of the colicin fragment into a bulge loop. S1 protein forms a strong 1:1 complex with the colicin fragment, with an association constant of 5 x 10(6) M-1 in 5 mM Mg2+. More protein molecules are bound, but with weaker affinity, when the S1 concentration is increased. S1 binding causes melting of the colicin fragment secondary structure, as inferred from the observed absorbance increase. The S1 binding site on the colicin fragment has been localized in the region of the bulge loop, since the melting transition corresponding to the 4-base pair helix is lost in the complex. We discuss current models for the role of S1 protein in polypeptide chain initiation in light of these and previous results.

Base Composition↗
PubMed1979

Characterization of a mutant form of ribosomal protein S1 from Escherichia coli.

An altered form of ribosomal protein S1 from a mutant of Escherichia coli has been isolated and characterized. The mutant protein (denoted m1-S1) has a molecular weight of 57,000 as shown by sodium dodecyl sulfate-gel electrophoresis and the same NH2-terminal sequence as wild type S1. Protein m1-S1 binds poly(U) in the same manner as protein S1 and is active in protein synthesis with either synthetic or natural mRNA. Thus, about 75% of the sequence of protein S1 (which includes the NH2-terminal region) contains essentially all the functional domains of this protein involved in protein biosynthesis.

Cross Reactions↗