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The deoxyoctanucleotide (5′-3′)d(A-A-G-G-A-G-G-T), which is complementary to the 3′ end of 16-S RNA, inhibits the formation of the complex between the 30-S subunit and MS2 RNA described in the preceding paper. If the complex is preformed, the octanucleotide cannot prevent entry of the complex into the ribosome cycle upon supplementation with the components for protein synthesis. The subunit · MS2-RNA complex is unable to bind the octanucleotide. It is concluded that in the subunit · phage-RNA initiation precursor the 16-S terminus is base-paired with a complementary MS2 RNA sequence. Edeine, aurintricarboxylic acid and antibodies against ribosomal protein S1 prevent the association of phage RNA with 30-S subunits. These compounds do not, however, inhibit the binding of (5′-3′)d(A-A-G-G-A-G-G-T) to 30-S subunits. It is concluded that the formation of the complex between MS2 RNA and 30-S subunits does not depend solely on the Shine and Dalgarno base-pairing reaction. [ABSTRACT FROM AUTHOR]

UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells. Furthermore, the attenuator model can only account for the results if it is assumed that UTP-concentration dependent pausing of transcription occurs in vivo in the two pry gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions. [ABSTRACT FROM AUTHOR]

The basic assumption in this paper is that the secondary structure era 5-S ribosomal RNA cannot be represented by a single model. We propose that the molecule can adopt, at least within the ribosome, a series of slightly different structures of nearly equal stability. The different structures arise from the existence of ambiguous base-pairing opportunities in bulged helices and the adjacent interior loops. In cubacterial 5 S RNAs there is one such an area, in eukaryotic 5-S RNAs two such areas that can give rise to structural switches. We explain how a change in secondary structure in these areas may influence the relative orientation of the surrounding helices, in other words how bulges and interior loops may serve as articulations and give rise to a flexible tertiary structure. [ABSTRACT FROM AUTHOR]

The procedures developed earlier (Friedrich and Woolley, preceding paper in this journal) for probing electrostatic potential with the fluorescein label were applied to transfer RNA. By using tRNA species that contain chemically reactive bases we were able to label these bases with fluorescein derivatives and thus to 'map' the electrostatic potential around the molecule. Both the electrostatic potential and the fluorescence emission anisotropy data that were obtained at the same time could be understood in terms of the well-known, paradigmatic crystal structure of tRNAPhe. However, within the distribution of the various tRNA species, tRNAfMet appeared to occupy an extreme position, which suggests a relation between the conformation in solution and the initiation function of this molecule. Comparison with theoretical predictions by others of the electrostatic potential map of tRNA showed agreement in respect of trends, but the values of the potentials measured were orders of magnitude lower than predicted. This we attribute primarily to solvation. [ABSTRACT FROM AUTHOR]

The nucleotide sequence of wheat (Triticum aestivum) 5.8-S ribosomal RNA has been re-examined using partial chemical degradation with high-temperature sequencing gels and oligonucleotide analysis. The results clarify previously ambiguous residues and add two additional nucleotides, G127 and G135 to the sequence. Estimates of the secondary structure suggest that 5.8-S rRNAs of higher plants differ from previous examples in having more open G + C-rich and A + U-rich stems. S1 ribonuclease digestion was used to probe this secondary structure; the data generally support the 'burp gun' model proposed for all 5.8-S rRNAs but are also consistent with a more open A + U-rich stem. Diethyl pyrocarbonate reactivity was used to probe the topography of this RNA in wheat ribosomes. The results indicate that the G + C-rich and A + U-rich stems are accessible to chemical modification in the wheat ribosome and suggest that the A + U-rich stem undergoes a significant conformational change when the molecule associates into ribosomes. [ABSTRACT FROM AUTHOR]

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry ( Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide–adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco ( Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide–adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins. [ABSTRACT FROM AUTHOR]

Studies the efficiency and specificity of RNA-protein cross-linking in the 30S subunit of Escherichia coli ribosomes induced by low-intensity and high-intensity photons. Observations of biphotonic processes in the high-intensity photon environment; Efficiency of cross-linking is 20-100 higher upon two-step excitation compared with single-step excitation.

Presents data which show that the arrangement of tRNA in the ribosome depends not only on which of the two mRNA codons inside the ribosome binds tRNA but also on the functional and the structural state of the elongating complex. Advantages for the more precise and more detailed classification of the tRNA-binding sites of the ribosome; Interaction with the functional state.

Ribosomal protein methylation has been well documented but its function remains unclear. We have examined this phenomenon using an Escherichia coli mutant (prmB2), which fails to methylate glutamine residue number 150 of ribosomal protein L3. This mutant exhibits a cold-sensitive phenotype: its growth rate at 22 °C is abnormally low in complete medium. in addition, strains with this mutation accumulate abnormal and unstable ribosomal particles; 50-S and 30-S subunits are formed, but at a lower rate. Once assembled, ribosomes with unmethylated L3 are fully active by several criteria. (a) Protein synthesis in vitro with purified 70-S prmB2 ribosomes is as active as wild-type using either a natural (R17) or an artificial [poly(U)] messenger. (b) The induction of β-galactosidase in vivo exhibits normal kinetics and the enzyme has a normal rate of thermal denaturation. (c) These ribosomes are standard when exposed in vitro to a low magnesium concentration or increasing molarities of LiCl. Efficient methylation of L3 in vitro requires either unfolded ribosomes or a mixture of ribosomal protein and RNA. We suggest that the L3-specific methyltransferase may qualify as one of the postulated 'assembly factors' of the E. coli ribosome. [ABSTRACT FROM AUTHOR]

A consensus on the folding of the Escherichia coli 16-S ribosomal RNA is emerging and several complete nucleotide sequences of small ribosomal subunit RNAs, covering diverse types of organisms and organelles, are now available. We therefore investigated the extent of both nucleotide sequence and secondary structure conservation that may exist between the E. coli 16-S RNA and other ribosomal RNAs. All the RNA molecules examined could be folded into secondary structure schemes that illustrated remarkable preservation of many structural motifs as well as striking nucleotide sequence conservation compared with the E. coli molecule. This study presents a unitary scheme for the structural organization of the small ribosomal subunit RNAs. The evolutionary constraints on both primary and secondary structures most likely reveal the basic role of some restricted RNA regions in the function of the ribosome. [ABSTRACT FROM AUTHOR]

Escherichia coli ribosomal protein S1 and its mutant, shorter, form m1-S1 were cleaved at internal methionyl residues to yield, respectively, six and five fragments of M1 ranging from 1000 to 24000. Methods are described to isolate the fragments in pure form. Four of the fragments (designated F2a, F2b, F3 and F4) contain between 86 and 215 amino acids and are therefore as large as other ribosomal proteins. Fragment F2a, derived from the N-terminal region, has previously been shown to contain the major ribosome binding domain of S1 [S. Giorginis and A. R. Subramanian (1980) J. Mot Biol. 1411 393 -408]. Here we show that the RNA binding domain of S1 is essentially contained in F3 derived from the middle region of S1 and carrying the nonreactive - SH group. The reactive a SF! group of SI, whose activity is modified by ligand binding, was localized in F2b, a fragment with little RNA binding capacity. The characteristic RNA binding domain and a weak ribosome binding domain of S1 have previously been localized in the large trypsin-resistent core SI-Fl [T. Suryanarayana and A. It Subramanian(1979) J. Mol. Biol. 127, 41-54]. Together these data indicate that two of the key functional domains of SI are located in two regions of the molecule separated by an open, exposed segment. The present study also revealed that the nonreactive - SM group of S1 becomes reactive in m1-S1 by the loss of the remote C-terminal region in the latter. [ABSTRACT FROM AUTHOR]

Crosslinking between phenylalanyl transfer RNA from brewer's yeast and poly(U) can be obtained when ribosomal complexes are irradiated at »300 nm; the ribosomal complexes were formed by enzymatic binding of the tRNA. The crosslinked product was isolated on oligo(dA)-cellulose columns. The experiments suggest that the photo-crosslinking occurs via the wybutine in the anticodon loop of the tRNA. This system can be used to study interactions between tRNA and messenger RNA on the ribosome. [ABSTRACT FROM AUTHOR]

Mycelia of Neurospora crassa in a steady state of growth in different media have a ribosomal content proportional to the rate of growth. Moreover, both the percentage of polysomes and the average ribosomal activity are about the same at all different growth rates. The content of poly- adenylated RNA was determined in three different conditions of exponential growth, which allowed growth rates that ranged from 0.26 to 0.51 duplications/h, and was found to constitute about the same fraction of total RNA (4.5–5.2 %). Using a kinetic approach, an equation was derived which allowed determination of the average half-lives of polyadenylated RNA: in each medium the cultures were labeled from the moment of the inoculation with [32P]orthophosphate and were then given a 10-min pulse with [5-3H]uridine when they were in the exponential phase. It was found that the determined half-lives of polyadenylated RNA vary, depending on the growth medium, between 30 and 60 min, but with no direct correlation with the growth rate. Moreover, the rate of synthesis of polyadenylated RNA relative to that of stable RNA decreased with the growth rate. On the basis of previous data on the rates of synthesis of stable RNA, it was possible to make an evaluation of the absolute rate of synthesis of polyadenylated RNA. Whereas the rate of synthesis of stable ribosomal RNA increases as a function of the square of the number of duplications per hour, the increase in the rate of synthesis of polyadenylated RNA with the growth rate is much less consistent. It is concluded that in Neurospora the growth rate does not depend on the rate of synthesis of mRNA but rather on the rate of synthesis of rRNA, which sets both the ribosomal level and the steady-state level of mRNA. [ABSTRACT FROM AUTHOR]

Incorporation of mannose into endogenous lipid and protein acceptors of the rat liver endoplasmic reticulum was investigated by incubating rough microsomes with GDP-[14C]mannose in the absence of detergent. Labeled dolichyl monophosphate mannose was readily Formed from the endogenous dolichyl monophosphate. in contrast, the label was not detectable in dolichyl diphosphate oligosaccharides and it was incorporated into protein only to a small extent, except when preparations previously treated with pyrophosphate were incubated in the presence of UDP-N-acetylglucosamine and GTP. Then, incorporation of label into such lipid and protein derivatives was considerably enhanced. GTP could not be substituted by other nucleotides. The requirement for UDP-N-acetyl-glucosamine, and other experimental evidence, demonstrate that mannosylated oligosaccharides which contain internal N-acetylglucosamine units are entirely assembled on dolichyl diphosphate from the exogenous nucleotide sugars. The kinetics of mannose incorporation into dolichol-linked oligosaccharides and into protein suggest that the protein acceptors are mainly mannosylated through transfer of oligosaccharides preassembled on dolichyl diphosphate. In heavy microsomes which have not been treated with PPi-or upon incubation in the absence of GTP, the amount of dolichyl phosphate which can accept N-acetylglucosamine 1-phosphate is less than that which can accept mannose. However, when PPi-treated preparations are incubated with UDP-N-acetylglucosamine and GDP-mannose in the presence of GTP, a larger amount of dolichyl phosphate is mobilized in the form of various saccharide derivatives of dolichyl diphosphate, and the amount of dolichyl monophosphate mannose is correspondingly decreased. It seems, therefore, that the membranes contain a particular pool of dolichyl monophosphate, which readily forms dolichyl monophosphate mannose, but reacts with UDP-N-acetylglucosamine only under appropriate conditions. This pool could act as precursor of the glycoside derivatives of dolichyl diphosphate which transfer their saccharide moiety to proteins. [ABSTRACT FROM AUTHOR]

Isolated rabbit reticulocyte ribosomes were treated with the toxic lectin modeccin for increasing periods of time. The toxin treatment was stopped by adding specific antitoxin and the residual activity of the ribosomes was tested in a polyphenylalanine-synthesizing system. With increasing time of toxin treatment the ability of the ribosomes to support polymerization of phenylalanine decreased. Addition of anti-toxin immediately stopped further inactivation of the ribosomes. The presence of elongation factor 2 (EF-2) strongly increased the rate of ribosome inactivation by modeccin. There was no similar effect with EF-1. Maximal rate of inactivation was observed when the molar ratio of EF-2 to ribosomes was about 1. The rate of ribosome inactivation was about 4 ribosomes min' (molecule of modeccin)' under optimal conditions. The ability of EF-2 to sensitize ribosomes to modeccin was strongly descreased in the presence of GTP, GDP and guanosine 5' -[βγ-methylene]triphosphate. EF-2, which had been ADP-ribosylated in the presence of diphtheria toxin, was fully active in sensitizing the ribosomes whereas N-ethylmaleimide-treated EF-2 had lost this ability. Isolated 60-S subunits were sensitized to modeccin by EF-2 in a similar way as whole ribosomes. The data indicate that modeccin inactivates ribosomes catalytically and that in the absence of nucleotides it binds to the 60-S subunit in a specific way. The possibility of two binding sites for EF-2 on the ribosomes is discussed. [ABSTRACT FROM AUTHOR]

The inactivation of rabbit reticulocyte ribosomes by abrin and ricin A-chains was studied by incubating ribosomes with the A-chains and testing, after various periods of time, aliquots of the The presence of elongation factor 2 (EF-2) reduced the rate of inactivation of ribosomes by the A-chains. The protective effect of EF-2 was strongly enhanced by GTP and, to a lesser extent, also by GDP or dGTP. Other nucleotides had no demonstrable effect. Much less protection was found after binding of Phe-tRNA to ribosomes in the presence of EF-1 (enzymic binding) or in the presence of high Mg2+ concentration (non-enzymic binding). The data indicate that when EF-2 binds to the ribosomes it completely or partially covers the target site for abrin and ricin A-chains. The possibility that EF-1 also binds to this site is discussed. [ABSTRACT FROM AUTHOR]

16-S ribosomal RNA and a complex of ribosomal protein S4 and 16-S rRNA were studied in solution by small-angle X-ray scattering. Concentration series of the 16-S rRNA and the S4 · 16-S-rRNA complex were measured in 37.5 mM Tris-HCl buffer pH 7.4 at 5°C. The following data were determined. The radii of gyration for the 16-S rRNA and S4 · 16S-rRNA complex were R = 17.6 ± 0.4 nm and 17.5 ± 0.6 nm, respectively. The two respective values of the radii of gyration of the cross-section were Rq.1 = 8.42 ± 0.1 nm and 8.33 ± 0.3 nm, and Rq.2 = 0.988 ± 0.03 nm and 0.996 ± 0.03 nm. The largest diameters of the 16-S RNA and S4 · 16-S-RNA complex were L = 61.8 ± 1 nm and 60.0 ± 1 nm, respectively. Volumes of V = 1570 ± 60 nm³ were found for both particles. In the Tris buffer used, no significant differences were found between the scattering curves of 16-S rRNA and the S4 · 16-S-rRNA complex. A model equivalent in scattering properties to the RNA and complex is a flat elliptical cylinder with the following dimensions: large axis 61.7 nm, small axis 35.4 nm and height 2 nm. The theoretical scattering curve fits the experimental one as long as the shape of the measured curve is due to the overall shape of the particle. A model equivalent in scattering over the whole measured angular range is one built up from a large numbe of spheres that simulate the known substructure of the RNA. The outer dimensions of this model correspond to those of the flat elliptical cylinder. [ABSTRACT FROM AUTHOR]

Limited digestion of the Escherichia coli 50-S subunit permits the isolation of two large fragments of the 23-S RNA. One arises from the 5' end of the 23-S RNA and contains about 1200 nucleotides. The other arises from the 3' end of the 23-S RNA and contains about 2000 nucleotides. Each of the ten 50-S proteins known to bind specifically and independently to the 23-S RNA were tested for their ability to interact with these two RNA fragments. It was determined that the 5' terminal segment contains the specific binding sites for proteins L4, L20 and L24, whereas the 3' terminal segment contains the specific binding sites for proteins LI, L2, L3, L6, L13, L16 and L23. [ABSTRACT FROM AUTHOR]

Titration of isolated Escherichia coli ribosomal 30-S particles with [14C]phenylalanyl-tRNA in the presence of poly(uridylic acid) was used for a quantitative assay of codon-dependent binding of aminoacyl-tRNA with the small ribosomal subparticle. The technique has allowed the estimation both of the fraction of "active" 30-S subparticles capable of forming the 30-S · poly(U) · phenylalanyl- tRNA complexes and the equilibrium constants of phenylalanyl-tRNA binding in different media. Heterogeneity of the ternary complexes formed has been revealed: at least two classes of complexes differing in stability have been observed. The stability of the 30-S · poly(U) · phenylalanyl-tRNA complexes has been shown to decrease with the lowering of the Mg2+ concentration, the increase of K+ concentration and the addition of urea. The stability of the complexes increases with the increase of Mg2+ concentration, with the addition of ethanol and decrease of temperature. It is demonstrated that the fraction of actively binding 30-S particles also varies in different medium conditions; it decreases with the increase of ionic strength (K+) and with the addition of urea, and increases with the increase of Mg2+ concentration and addition of ethanol. [ABSTRACT FROM AUTHOR]

Using equilibrium dialysis techniques it could be demonstrated that (a) the pleuromutilin derivative 14-deoxy-14[(2-diethylaminoethyl)-mercaptoacetoxy] dihydromutilin HCl binds to one site per ribosome specifically, (b) the binding constant is 1.3 × 107 M-1 and (c) chloramphenicol and puromycin compete with binding of the pleuromutilin derivative. Similarly the nudeotides CpA and CpCpA also displace the unsaturated derivative of the above-mentioned pleuromutilin compound. These findings suggest that the ribosomal binding site for pleuromutilin overlaps with that for chloramphenicol and analogs of the 3'-terminus of a tRNA, like puromycin or the nudeotides CpA and CpCpA. [ABSTRACT FROM AUTHOR]

Tritiated elongation factors 1 and 2 (EF-1 and EF-2) were obtained from Krebs II ascites cells which had been grown in mice injected with radioactive amino acids. The highly purified factors were sufficiently radioactive to be used in a study of the interactions between ribosomes and elongation factors. The following results were obtained. 1. EF-1 binding to ribosomes requires the presence of a polynucleotide, an aminoacyl-tRNA specified by the latter and a guanosine nucleotide carrying three phosphate groups. The hydrolysis of the GTP molecule involved in the binding reaction leads to the immediate release of EF-1. If GTP is replaced by Guo-5'-P2-CH2-P the factor remains bound to the ribosome and can be detected by sucrose gradient centrifugation techniques. 2. Likewise EF-2 binding to ribosomes can only be detected in the presence of Guo-5'-P2-CH2-P. 3. The affinity of ribosomes for EF-2 appears to be higher than for EF-1: preincubation of ribosomes with EF-2 inhibits the subsequent attachment of EF-1 almost completely. EF-1 prebound to ribosomes in the presence of Guo-5'-P2-CH2-P, poly(uridylic acid) and Phe-tRNAPhe is partially removed from the ribosomes together with Phe-tRNA during a second incubation with EF-2. 4. Although EF-2 binding to ribosomes precludes any stable association between ribosomes and EF-1 it does not prevent the insertion of aminoacyl-tRNA into the ribosomal A-site. The attachment of aminoacyl-tRNA under these conditions enhances the binding of EF-2 to the ribosome. 5. The antibiotic showdomycin strongly inhibits the attachment of EF-1 to ribosomes and to a lesser degree impairs the binding of EF-2. 6. A-site ribosomes display a strong preference for the attachment of EF-2 and bind EF-1 only very poorly. The reverse is true for P-site ribosomes which are good substrates for the binding of EF-1 and bind EF-2 less efficiently than A-site ribosomes. These results and a number of additional findings made in this and in previous studies are discussed in the general context of the structure and function of mammalian elongation factors 1 and 2. [ABSTRACT FROM AUTHOR]