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In this paper the question was studied whether Escherichia coli ribosomes which have undergone a specific cleavage of their 16-S rRNA by treatment with the bacteriocin cloacin DF13, are able to enter and complete an elongation cycle. As we have shown in the preceding paper in this journal, these defective ribosomes can form 70-S initiation complexes with MS2 RNA as a messenger. Binding of the second aminoacyl-tRNA (alanyl-tRNA) to the latter complexes led to the formation of fMet-Ala-tRNA which was fully puromyein-sensitive. In addition to these aminoacyl-tRNAs also the third aminoacyl-tRNA (seryl-tRNA) can be bound to defective ribosomes. Dual-label experiments showed that puromycin released all radioactivity from the ribosomes. This indicates that fMet-Ala-Ser-tRNA is formed and is translocated from the A site to the P site. Control ribosomes bind the three aminoacyl-tRNAs in a 1:1:1 ratio. The defective particles bind fMet-tRNA, Ala-tRNA and Ser-tRNA in a ratio of 1:0.5:0.25. Kinetic experiments strongly suggest that at each binding of the ternary complex aminoacyt-tRNA · EF-Tu · GTP to the A site of defective ribosomes, about half of these particles undergo irreversible inactivation. Consequently polypeptide synthesis under our conditions will come to an end after incorporation of only four or five amino acids. The particles which temporarily survive ternary complex binding are fully capable of transpeptidation and translocation. [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]

We show in the present paper that the cleavages initiating decay of the ompA mRNA are suppressed both in the Escherichia coli amsts strain (originally defined by a prolonged bulk mRNA half-life) and in the rnets strain (originally defined by aberrant 9S RNA processing). The temperature-sensitive defects of both these strains are complemented by a recombinant lambda phage containing a genomic segment that carries the putative ams locus. A 5.8kb fragment from this genomic DNA segment was cloned into a low-copy plasmid and used to transform the amsts and rnets strains. This resulted in growth at the non-permissive temperature and a reoccurrence of the cleavages initiating decay of the ompA mRNA. Deletion analyses of this 5.8 kb fragment indicated that the putative ams open reading frame could complement both the Amsts and the Rnets phenotype with regard to the ompA cleavages. In addition we showed that the amsts strain suppresses 9S RNA processing to 5S RNA to the same extent as the rnets strain, and that the rnets strain has a prolonged bulk mRNA half-life, as was reported for the amsts strain. Therefore we suggest that ams and rne reflect the same gene locus; one which is involved both in mRNA decay and RNA processing. We discuss how this gene locus may related to the previously characterized endoribonucleolytic activities of RNase E and RNase K. [ABSTRACT FROM AUTHOR]

The complete nucleotide sequence of the 16-S RNA from Escherichia coli has been determined using rapid RNA-sequencing gel methods. The experimental data are fully described in this paper. The specificities of the ribonucleases, especially the ribonuclease PhyI are discussed and the consequences of the persistence of stable secondary structure are considered. The proposed sequence contains 1541 nucleotides and agrees completely with the DNA sequence of the rrnB cistron deduced by Brosius, J., Palmer, M. L., Kennedy, P.J., and Noller, H. F. [Proc. Natl Acad. Sci. U.S.A. (1978) 75, 4801–4805]. But there are several cistron heterogeneities of which we described 16 single-base heterogeneities, 7 of the deletion/insertion type and 9 of the transition or transversion type. Our observations suggest the existence, among the 7 ribosome RNA cistrons, of one or two mutated ones. The respective advantages and disadvantages of both RNA and DNA sequencing methods are discussed. [ABSTRACT FROM AUTHOR]

When Escherichia coli 30-S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub-fragments have therefore been examined by oligonucleotide analysis. Two well-defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed. The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub-fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5′-terminal 900 nucleotides of 16-S RNA, corresponding to the well-known 12-S fragment. No long-range interactions could be detected within this RNA region in these experiments. The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9, S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3′ end of the 16-S RNA, but lacking the 3′-terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16-S RNA, but section J (the 3′-terminal 50 nucleotides) was not found. This result suggests that the 3′ region of 16-S RNA is not involved in stable interactions with protein. [ABSTRACT FROM AUTHOR]

The two types of 30-S · 50-S couples described in the preceding paper, differ markedly in their sensitivity to hydrostatic pressure evoked during centrifugation in sucrose gradients. The sedimentation coefficient of about 60 S recorded with couples formed from native subunits is only apparent, the real a value being 70 S. The latter couples can be stabilized against pressure-induced dissociation by bound tRNA. On the other hand this binding renders couples formed from derived subunits more sensitive to this dissociation. As the factor determining this intriguing difference in pressure sensitivity lies in the 50-S subunit, a comparative analysis of a number of ribosomal constituents has been performed for native and derived 50-S particles. No qualitative differences in the proteins L1–L34 have been detected. Quantitative differences cannot yet be excluded. It is considered unlikely that differences in rRNAs or low-molecular-weight components arc responsible for the distinct association behaviour. Partial reconstitution of ribosomal γ-cores and split proteins detached by CsCl reveals that the determinant factor is located in the core particles. This suggests that native and derived 50-S subunits do not differ in conformation only. The possibility may be envisaged therefore, that the two types of 50-S differ in a ribosomal protein. [ABSTRACT FROM AUTHOR]

It is well know that protein synthesis stopped either by chloromycetin or by starvation of an essential amino acid for a RC relstrain, ribosomal RNA synthesis continues, but products do not appear in 50 S and 30 ribosomes. It has also been shown that 5 S RNA synthesis is altered by the addition of chloromycetin but in an unknown manner. In this paper we report that: (a) during amino-acid starvation of a starvation of RCrel strain of Escherichia coli, 5 S RNA is transcribed as 5 S relax RNA located outside the ribosomes, in the supernatant fraction; (b) this 5 S RNA which is slightly longer than the normal product is convertible into a true 5 S RNA when protein synthesis is restored after blockage of RNA synthesis by actinomycin ;(c) part of the 23 S RNA from a 25 S particle appears after resumption of protein synthesis in a 40 S parcel which contains normal 5S RNA; (d) a pool of 5 S precursor RNA exists outside the 50 S ribosome. This 5 S precursor RNA is isolated; (e) this precursor which is isolated from the 105000xg supernatant fraction after pulse labeling, migrates on gel electrophoresis between 5 S relax RNA and true 5 S RNA. [ABSTRACT FROM AUTHOR]

The crystal structure of Thermus thermophilus ribosomal protein TL5 in complex with a fragment of Escherichia coli 5S rRNA has been determined at 2.3 Å resolution. The protein consists of two domains. The structure of the N-terminal domain is close to the structure of E. coli ribosomal protein L25, but the C-terminal domain represents a new fold composed of seven β-strands connected by long loops. TLS binds to the RNA through its N-terminal domain, whereas the C-terminal domain is not included in this interaction. Cd2+ ions, the presence of which improved the crystal quality significantly, bind only to the protein component of the complex and stabilize the protein molecule itself and the interactions between the two molecules in the asymmetric unit of the crystal. The TLS sequence reveals homology to the so- called general stress protein CTC. The hydrophobic cores which stabilize both TLS domains are highly conserved in CTC proteins. Thus, all CTC proteins may fold with a topology close to that of TL5. [ABSTRACT FROM AUTHOR]

A resistance gene, carB, originally isolated from the carbomycin-producing organism. Streptomyces thermotolerans, confers on Streptomyces lividans high-level resistance to the drug. However, ribosomes from S. lividans expressing carB show only moderate resistance to this macrolide in vitro, although they are highly resistant to the action of lincosamide antibiotics. The carB product monomethylates the amino group of the adenosine residue located at position 2058 in 23S ribosomal RNA. In contrast, ribosomes from S. lividans expressing ermE, in which 23S RNA is dimethylated at this same position, are much more highly resistant to macrolides and insensitive to lincosamides. [ABSTRACT FROM AUTHOR]

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-S RNA in the absence of ribosomal protein. These data yield important information on the topography and organization of the 5′ third of the 16-S RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA. [ABSTRACT FROM AUTHOR]

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with out previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A, 73, 702–706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can he compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subnnit interaction. [ABSTRACT FROM AUTHOR]

The primary structures of the 16S and 23S ribosomal RNAs of Escherichia coli have been studied. Nucleotide sequences have been determined which occur in specific areas close to the small numbers of methylated nucleotides which are present in these molecules. Fragments containing these methylated nucleotides were obtained by digestion of radioactive RNA substrates with T1 ribonuclease. Such digestion products were fractionated by high-voltage electrophoresis in two dimensions. The 23S RNA yielded predominantly 11 methylated oligonucleotides on digestion, and the 16S RNA gave rise to 6 methylated components. Nucleotide sequence analysis of these products was carried out, and the nature of the modified nucleotides occurring in each product was also examined. It was found that some of the sequences contained several modified components positioned close to each other. Quantitative studies indicate that each of the methylated sequences is probably present twice in a molecule of the 23S RNA, so that these sequences encompass about 5% of the nucleotides present in the entire molecule. It is postulated that extensive repetition of nucleotide sequences occurs within the 23S RNA. [ABSTRACT FROM AUTHOR]

RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level. [ABSTRACT FROM AUTHOR]

In Escherichia coli strains carrying null mutations in either the dnaK or dnaJ genes, the late stages of 30S and 50S ribosomal subunit biogenesis are slowed down in a temperature-dependent manner. At high temperature (44°C), 32S and 45S particles (precursors to 50S subunits) and 21S particles (precursors to 30S subunits) accumulate. The latter are shown by 3′5′ rapid amplification of cDNA ends analysis to contain unprocessed or partially processed 16S ribosomal RNA at the 5′ end, but the 3′ end was never processed. This implies that maturation of 16S ribosomal RNA starts at the 5′-terminus, and that the 3′-terminus is only trimmed at a later step. At normal temperatures (30°C−37°C), ribosome assembly in both mutants is not arrested but is significantly delayed, as shown by pulse-chase analysis. Assembly defects are partially compensated by an overexpression of other heat-shock proteins, which occurs in the absence of their negative regulator DnaK, or by a plasmid-driven overexpression of GroES/GroEL, suggesting the involvement of a network of chaperones in ribosome biogenesis. [ABSTRACT FROM AUTHOR]

In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA. While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3′ and 5′ termini. We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product. Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates. When both RNase E and CafA are inactivated, 5′ maturation of 16S rRNA is completely blocked. In contrast, 3′ maturation is essentially unaffected. The 5′ unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes. Precursors also can be processed in vitro by RNase E and CafA. These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5′ end of 16S rRNA, and that CafA protein is a new ribonuclease. We propose that it be renamed RNase G. [ABSTRACT FROM AUTHOR]

The structure of the Escherichia coli ribosomal protein L25 has been determined to an r.m.s. displacement of backbone heavy atoms of 0.62 ± 0.14 Å by multidimensional heteronuclear NMR spectroscopy on protein samples uniformly labeled with 15N or 15N/13C. L25 shows a new topology for RNA-binding proteins consisting of a six-stranded β-barrel and two α-helices. A putative RNA-binding surface for L25 has been obtained by comparison of backbone 15N chemical shifts for L25 with and without a bound cognate RNA containing the eubacterial E-loop that is the site for binding of L25 to 5S ribosomal RNA. Sequence comparisons with related proteins, including the general stress protein, CTC, show that the residues involved in RNA binding are highly conserved, thereby providing further confirmation of the binding surface. Tertiary structure comparisons indicate that the six-stranded β-barrels of L25 and of the tRNA anticodon-binding domain of glutaminyl-tRNA synthetase are similar. [ABSTRACT FROM AUTHOR]

We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the α-sarcin family of ribosome-inactivating proteins (RIPs). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2β) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates. [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.

The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible λ PL promoter. After induction of transcription by thermal inactivation of the λ ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10°C, but poor expression was obtained if the cells were incubated at 37°C or 30°C. The reason for this differential temperature-dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37°C compared to 10°C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half-lives of the cspA transcript were substantially longer at 10°C compared to 37°C; (iii) S30 extracts as well as 70S ribosomes prepared from cold-shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37°C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold-shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock. [ABSTRACT FROM AUTHOR]

We have been able to isolate several species of 5-S ribosomal RNA from Eschzerichia coli A19. These molecules were separated on the basis of their differing stabilities during electrophoresis on 12% polyacrylamide gels in 7 M urea. This differing stability is shown, in one case, to be due to a different primary sequence. We have determined the sequence of the least stable of these molecules and have found only one difference to the published sequence of E. coli A19 5-S RNA, namely a uridine in place of a cytidine at position 92. The consequent G U base pair, formed in a normally highly stable G. C-rich region. is responsible for a drastic reduction in the stability of the molecule. This instability leads to a less constrained, more compact molecule which thus migrates laster in electrophoresis under denaturing conditions. This species of 5-S RNA is shown to make up 30% of the total 5-S RNA in the 50-S ribosomal subunits in this organism. Further structural studies were carried out using S1 nuclease digestion, sodium bisulphite modification and thermal melting analysis. All these methods indicate a 5-S RNA drastically destabilized in parts of its secondary and tertiary structure. Finally, the ability of the variant 5-S RNA to recognize and form a complex with its 50-S subunit binding proteins was examined and found to be impaired. [ABSTRACT FROM AUTHOR]

Modification of the 50-S subunits of Escherichia coli ribosomes with the arginine reagent phenylglyosal produces inactivation of peptidyl transferase and inhibition of the binding of C(U)-A-C-C-A-LeuAc, phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA to the ribosome. Hybridization experiments, using 1.25 M LiCl core particles and the corresponding split proteins from untreated and phenylglyosal-treated 50-S subunits, indicate that inactivation and inhibition of binding are the effects of modification of a protein fraction, the functionality of the RNA moiety being unaffected by the reagent. The split proteins from phenylglyosal-modified 50-S subunits are incorporated to 1.25 M LiCl core particles as well as those obtained from unmodified subunits, thus excluding the failure to bind as the cause of inactivation. In agreement with the general role played by the arginyl residues as positive binding sites for anionic ligands, the present results indicate that the arginyl residues of a protein fraction from 50-S subunits be important in the binding of aminoacyl-tRNA and peptidyl-tRNA to ribosomes. [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]

Analysis of 16-S rRNA synthesized in Eseherichia coli D10 (met-) incubated in a medium containing ethionine in place of methionine shows that it lacks most and probably all of the methyl groups present in normal 16-S rRNA but possesses the same 3'-OH, and 5′-phosphate terminal sequences as the latter. 23-S rRNA formed in ethionine-treated cells also contains normal terminal sequences. 5-S rRNAs of normal and ethionine-treated E. coli D10 are identical. These results lead to the conclusion that methylation of ribosomal precursor RYAs is not necessary for their maturation to products with normal chain lengths and does not influence the conformation of 16-S rRNA.. [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]

Comparison of the properties in vitro of total 30-S ribosomal subunit proteins and purified protein S4 of Escherichia coli D10 (wild type) and E. coli 219tS2 (temperature-sensitive) has given the following results. Reconstitution of functional 30-S subunits hi vitro occurs when total 30-S subunit proteins of either strain are used even at temperatures which are not permissive for 30-S subunit assembly in vivo in E. coli 219ts2. The yield of reconstitution is, however, twofold lower with 30-S subunit proteins of E. coli 219ts2 than with wild-type proteins. The yield of complex formation between 16-S rRNA and protein S4 of E. coli 219ts2 is temperature-sensitive and lower at all temperatures tested (33–42 °C) than that observed when wild-type S4 is used. The conformational stability of complexes between 16-S rRNA and S4 from 219ts2 is more temperature-sensitive than that of analogous complexes containing wild-type S4. These observations provide an explanation for the temperature sensitivity of 30-S subunit assembly in E. coli 219ts2. [ABSTRACT FROM AUTHOR]

Analysis of various RNA species from Escherichia coli. The RNAs were prepared under mild conditions without spreading using the shadow-cast technique. The 16-S RNA looks like a flattened balloon of diameter 18 ± 1.9 nm with a smooth surface and regular contours. In contrast, structural domains can be detected in 23-S RNA; typically a central domain is surrounded by five to seven satellite domains, with an overall diameter of 33 ±_ 4.5 nm. Different 23-S RNA preparations, which also behave differently in reconstitution assays, differ with respect to extent and height of the satellite structure. 5-S RNA shows an extended rod-like structure with a length of 11 ± 1.6 nm. A more corpuscular and prominent structure was observed for tRNA, with diameters Phe of 7.7 ± 1.1 nm and 8.9 ± 1.0 nm in case of tRNAPhe, and N-acetylphenylalanyl-tRNAPhe respectively. Analysis of the assembly in vitro of the ribosomal 50-S subunit. The assembly of the 50-S subunit in vitro occurs via three reconstitution intermediates, which are formed subsequently during the first incubation of the two-step reconstitution procedure [Dohme, F. and Nierhaus, K. H. (1976) J. Mol. Biol. 107, 585-599]. A total reconstitution was performed, aliquots were withdrawn during the first incubation at various times and analyzed in the electron microscope. A second aliquot was taken at the same times, subjected to a standard second incubation and tested for poly-(Phe)-synthesizing activity in the presence of 30-S subunits. The activity obtained is a measure of the amount of (second plus third) reconsitution intermediates present in the aliquot. With this parameter it was possible to select a series of subsequent assembly states from the electron micrographs which cinematographically illustrate the assembly of the 50-S subunit in vitro. The results indicate that the reconstitution intermediate stage RI[IPQ]50(1) comprises various particles, which evidently are formed consecutively. In contrast, the following reconstitution intermediate stage RI50(2) appears as a homogeneous population of particles. [ABSTRACT FROM AUTHOR]

The effects of ultraviolet irradiation on Escherichia coli 30-S ribosomal subunits were studied. At the doses of radiation used in this work (0–4.5 ⊗ 105 quanta/30-S subunit), only protein S7 was found to be significantly crosslinked to the 16-S RNA. In conditions where 25% of the protein was covalently crosslinked, the ability of the irradiated 30-S subunits to reassociate with 50-S subunits and their activity in polyphenylalanine synthesis decreased strongly. Similar results were obtained by irrradiation with a germicide lamp (254 nm) or with a monochromatic ultraviolet light at 248 nm. No additional proteins were crosslinked to the 16-S RNA by irradiating 30-S subunits depleted in protein S1 or 70-S ribosomes. The covalent complex of 16-S RNA and protein S7 was isolated and digested by T1 ribonuclease. The oligonucleotide remaining attached to the crosslinked protein was characterised as A-C-C-U-C-G [position 1261–1266, see the sequence published by Carbon et al. (1979) Eur. J. Biochem. 160, 399–410]. Analysis of this fragment suggests that protein S7 was linked to the cytosine at position 1265 in the RNA sequence. [ABSTRACT FROM AUTHOR]

Substantial evidence is provided to corroborate our previous finding that Escherichia coli ribosomes recognize two binding sites on the 5′ end of alfalfa mosaic virus (AMV) RNA 4 [for a preliminary report see Castel, A., Kraal, B., Kerklaan, P. R. M., Klok, J., and Bosch, L. (1977) Proc. Natl Acad. Sci. U.S,A. 74, 5509–5513]. Translation can start at either site using AcPhe-tRNA or fMet-tRNA as initiator and takes place in the same reading frame along the monocistronic mRNA. The size and composition of the isolated extra NH2-terminal fragment of the acetylphenylalanyl product were found to be in agreement with the 5′ non-coding region of the messenger. Removal of the 5′-terminal cap structure of AMV RNA 4 did not influence significantly both initiation reactions. Ribosomal protein S1 was essential for binding as well as incorporation of both fMet-tRNA and AcPhe-tRNA. A similar interaction on the ribosome was found for AcPhe-tRNA directed by AMV RNA 4 as for fMet-tRNA directed by either AMV RNA 4 or MS2 RNA with respect to the influence of initiation factors. It is concluded that the heterologous plant viral messenger is reliably translated in the E. coli system and that E, coli ribosomes recognize with high specificity an extra initiation site close to the 5′ extremity of the messenger. The relation- ship of this site to a hypothetical entry site involved in the early recognition in the initiation mechanism between ribosome and messenger is discussed. [ABSTRACT FROM AUTHOR]

The poly(U)-dependent binding of Tyr-tRNATyr to Escherichia coli ribosomes has been studied using a highly purified system. Binding is maximal at 10 mM magnesium acetate (up to 0.7 molecule Tyr-tRNATyr/ribosome), and requires the presence of elongation factor (EF) T (a mixture of EF-Ts and EF-Tu), GTP, NH4+ ions and an aminoglycoside antibiotic (streptomycin, neomycin B, kanamycip B or gentamicin C1a). Under limiting and up to saturating concentrations of EF-T, one molecule of GTP is hydrolyzed per molecule of Tyr-tRNATyr bound, suggesting that ‘proof-reading’ mechanisms involving the hydrolysis of GTP are inoperative in the presence of the antibiotics. Binding of Tyr-tRNATyr apparently takes place at the ribosomal acceptor site, since peptide bonds are readily formed with N-acetyl-Phe-tRNA prebound to the ribosomal donor site. In contrast to Phe-tRNAPhe binding, Tyr-tRNATyr binding is impaired by the omission of the 50-S subunit, the replacement of GTP by its non-hydrolyzable analogs guanyl-5′-yl methylene diphosphonate and guanyl-5′-yl iminodiphosphonate, and also by the presence of the antibiotic streptogramin A. This suggests that the correct interaction of Tyr-tRNATyr with the peptidyl transferase centre is essential for the stability of this ligand on the ribosome. Moreover, the aminoglycoside antibiotics are also necessary, even after the binding reaction is complete, to maintain Tyr-tRNATyr on the ribosome. [ABSTRACT FROM AUTHOR]

An affinity chromatographic procedure, using ribosomal or tRNA covalently coupled to agarose through an adipic acid dihydrazide spacer, has been developed to isolate and identify the Escherichia coli ribosomal proteins which specifically bind to these RNA molecules. Transfer RNA, 5-S, 16-S and 23-S ribosomal RNA were readily immobilized on agarose-dihydrazide; the maximum amount coupled decreasing with increasing molecular size. Specific binding of ribosomal proteins to RNA was observed in buffer containing 0.3 M KCl and 0.02 M MgCl2. Bound proteins were eluted with a high-salt, 2 M KCl, buffer containing 0005 M EDTA. Two ribosomal proteins. identified as L18 and L25 by two-dimensional gel electrophoresis, bound tightly to E. coli 5-S RNA; small amounts of a third protein, L5, also were bound. These proteins did not bind to immobilized tRNA nor did 30-S ribosomal proteins bind to 5-S RNA. Denatured 5-S RNA bound less L18 and L25 than the native species. Several 30-S and 50-S ribosomal proteins bind to immobilized tRNA. The major 50-S subunit proteins tightly bound to tRNA were identified as L3, L4, L5, L7 and L8/L9. Smaller amounts of proteins L1, L2, L11, L16 and L21 were also observed. Proteins L2 and L16 were retarded by agarose-bound tRNA. Protein S3 was the major 30-S subunit protein bound to tRNA. Lesser quantities of proteins S6, S9, S13 and S18 also interacted with tRNA. [ABSTRACT FROM AUTHOR]

32-P-labelled 50-S subunits from Escherichia coli ribosomes were hydrolysed under conditions known to give rise to two specific ribonucleoprotein fragments, containing proteins L1, L9, and L5, L18 and L25 respectively. RNA corresponding to these ribonucleoproteins was isolated and purified, and the various RNA fragments obtained were subjected to oligonucleotide analysis. The results showed that the RNA associated with proteins L1 and L9 was very similar to the RNA found with protein L1 after controlled digestion of 23-S-RNA · L1 complexes (described elsewhere); this RNA lies within a region 550–1000 nucleotides from the 3′ terminus of 23-S RNA. The RNA associated with proteins L5, L18, and L25 consisted predominantly of two species of similar size. One was 5-S RNA, and the other a fragment of 23-S RNA, lying within the region 450–1000 nucleotides from the 3′ terminus. [ABSTRACT FROM AUTHOR]

F2 phage RNA treated with O-methylhydroxylamine under denaturing conditions loses its ordered structure with consequent exposure of the normally hidden initiation codons. In the presence of Escherichia coli ribosomes and crude initiation factors modified f2 RNA binds about 50 times more f-[³H]-Met-tRNA than native f2 RNA. The interaction of native f2 [14C]RNA to E. coli 70-S or 30-S ribosomes does not depend on the presence of initiation factors. A significant number of ribosomes deficient in initiation factors interact with a molecule of modified f2 [14C]RNA. Treatment of the resultant polysomal complex with pancreatic Rnase yields ribosomes with f2 RNA fragments protected against Rnase. Almost all AUG/GUG codons in the f2 RNA are located on the Rnse-insensitive ribosome-bound fragments, constituting only 25% of the entire molecule. Addition of crude initiation factors to such ribosomes with fragments of modified f2 RNA promotes binding of f-[³H]Met-tRNA. The resultant complex is fully reactive with puromycin. No binding of Ac-Phe-tRNA takes place under similar conditions. [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]

The 3'-terminal sequences of 16-S ribosomal RNA from a number of bacteria have been determined by a stepwise degradation and 3'-terminal labelling procedure. The sequence obtained were: Bacillus stearothermophilus, -G(Z)≈5 Y-U-C-C-U-U-U-C-U (A); B subtilis, -G(Z)≈7 Y-C-U-U-U-C-U; Caulobacter crescentus, -G(Z)3 Y-U-C-C-U-U-U-C-U; Pseudomonas aeruginosa, -G-Z-Z-Y-C-U-C-U-C-C-U-U(A), where Z is any nucleotide other than G. Thus, as previously found in Escherichia coli, all bacterial 16-S rRNAs contain a pyrimidine-rich tract at the 3'-terminus. In B. stearothermophilus and Ps. aeruginosa this region shows substantial heterogeneity involving the 3'-terminal adenylic acid. A low level of 3'-terminal heterogeneity cannot be excluded for the other bacterial 16-S rRNAs examined. The 3'-termini of bacterial 16-S rRNA can be divided into two groups on the basis of sequence homology. The first group comprises E. coli and Ps. aeruginosa; the second, B. stearothermophilus, B. subtilis and C. crescentus. This division correlates with a previous separation of bacterial ribosomes into two categories based on ability to translate different mRNA preparations [Stallcup, Sharrock & Rabinowitz (1974) Biochem. Biophys. Res. Commun. 58, 92-98]. We have previously proposed that the precise base sequence at the 3'-terminus of 16-S rRNA determines the intrinsic capacity of bacterial ribosomes to translate a particular cistron [Shine & Dalgarno (1975) Nature (Lond.) 254, 34-38]. No difference was found in the 3'-terminal heptanucleotide sequence of 16-S rRNA from bacgteriophage T7-infected E. coli, as compared to that in uninfected cells. Thus, the T7-induced alteration in translational specificity of E. coli ribosomes is probably not mediated by modification of the terminal seven nucleotides of the smaller rRNA. The 3'-terminal sequences of the 23-S rRNA species were also determined. The sequences obtained were: B. stearothermophilus and B. subtilis, -Y-C; C. cresentus, -Y-C-U; Ps. aeruginosa, -Y-C-A; E. coli, -G-Y-U-U-A-A-C-C-U-U. No evidence for 3'-terminal heterogeneity was found. The results obtained are discussed in relation to possible base-pairing roles for the 3'-end of 16-S rRNA in bacterial protein synthesis. [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]

The RNA binding sites of four 30-S ribosomal subunit proteins from Escherichia coli, namely S4, S8, S15 and S20 were prepared from reconstituted single protein 16-S-RNA complexes by mild enzymic digestion of non-protected RNA regions. Oligonucleotide fingerprints of the protected RNA regions were obtained and their Positions were located within the 16-S RNA sequence. They were not completely contiguous regions of RNA; oligonucleotides had been excised from each of them. The binding sites of S4 and S20, and those of S8 and S15 showed overlapping. The specificity of the RNA binding sites was confirmed by a reconstitution method. [ABSTRACT FROM AUTHOR]

16-S RNA and five pure ribosomal proteins (S3, S4, S6, S7 and S8) from Escherichia coli 30-S subunits were studied by circular dichroism in regard to the reconstitution of biologically active subunits. A small structural variation upon heating could be measured for RNA. It cannot be interpreted in terms of stacking or disruption of hydrogen bonds. However, most proteins underwent important structural modifications when heated to 42 °C in reconstitution solvent. This effect was not dependent on the presence of magnesium salt. The relative amounts of secondary structure of native and heat-denatured ribosomal proteins were calculated by using three reference curves obtained from standard proteins, the exact secondary structure of which has been determined by X-ray crystallography. In contrast with earlier results, about 18 %, β-structure was detected in native proteins. This discrepancy can most probably be attributed to the calculation method used. Results are discussed in relation to reconstitution of ribosomes. [ABSTRACT FROM AUTHOR]

In Escherichia coli cells infected with the bacteriophage MS2, pronounced inhibition of ribosomal RNA synthesis occurs amounting to 50% of that of the uninfected control cells at 30 min post-infection. Over the same period the synthesis of tRNA, mRNA and total protein remains close to the level of uninfected E. coli, yet the synthesis of host protein is gradually replaced by that of viral origin. After 30-35 min post-infection the macromolecular synthesis of all the types under study significantly decreases. It is suggested that suppression of host protein synthesis is caused not by selective inhibition at transcription or translation level, but is rather a result of competition between E. coil mRNA and MS2 RNA. On the other hand, exclusive suppression of only ribosomal RNA synthesis may arise from changes in the regulation of transcription of ribosomal RNA locus.

Association between ribosomal subunits of Eschrichia coli can lead to the formation of two classes of 30-S · 50-S couples. The first class sedimenting between 50 S and 70 S, arises when native subunits associate, the second sedimenting at 70 S upon coupling of so-called derived subunits. From cross experiments it is concluded that it is the 50-S subunit which determines the nature of the association product, whereas the 30-S subunit determines whether an association product can be formed. Native and derived 50-S subunits are indistinguishable by sucrose gradient centrifugation and do not differ qualitatively in their ability to form polypeptides under the directions of bacteriophage RNA. Native 30-8 subunits lose a factor during isolation, probably IF-3. They are therefore able to associate both at 0° and 37 °C. On the other hand derived 30-S subunits are unable to associate at 0 °C but they do so at 37 °C in the presence of 10 mM Mg2+. A number of trivial factors have been excluded to explain the difference between the two types of 50-S subunits. The preparation method is not responsible for the difference. Nor do the derived subunits contain peptidyl-tRNA, known to influence subunit association. The content of tRNA and aminoacyl-tRNA has been determined quantitatively by polyacrylamide-gel electrophoresis. None of the subunits nor the 70-S ribosomes from which the derived subunits are prepared contain a tRNA species. [ABSTRACT FROM AUTHOR]

A fraction containing a complex between initiation factors F2 and F3 was isolated from Escherichia coli ribosomes. In contrast to the free factors, this F2 · F3 complex makes a stable ternary association with formylmethionyl-tRNA and GTP. F2 · F3 complex can be formed on the ribosomes during the process of initiation. It acts preferentially on the 30 S ribosomal subunit to which it confers a much larger affinity for fMet-tRNA than observed with the free factors. A model for the function of F2 · F3 complex as an intermediate in the initiation cycle is proposed. [ABSTRACT FROM AUTHOR]

The electrophoretic mobilities of mitochondrial and cell-sap ribosomal RNAs from Saccharomyces carlsbergensis have been measured relative to the mobility of Escherichia coli rRNA in 2.4% polyacrylamide gels at various temperatures and ionic strengths. At 5° C and in 20 sodium acetate, 40 mM Tris-acetate, 2 mM EDTA (pH 7.8), mitochondrial and cell-sap rRNAs co-migrated and were only partially resolved from E. coli rRNA. When the temperature of electrophoresis was increased the mobility of the mitochondrial rRNAs (and to a lesser extent of the cell-sap rRNAs) decreased relative to the mobility of the E. coli rRNA species, reaching a minimum between 9 and 16° C. As a consequence the mitochondrial and cell-sap rRNAs were completely resolved in this buffer at temperatures above 9° C. Similar effects of temperature on relative electrophoretic mobility were observed m 90 mM Tris-borate buffer at pH 8.3 and the effect was even more pronounced at low ionic strength (10 mM NaCl, 5 mM Tris-HCl, 2 mM EDTA, pH 7.5). At low ionic strength the decrease in the relative electrophoretic mobility of mitochondrial rRNA with an increase hi temperature was accompanied by a decrease in sedimentation coefficient from 21-22 S (large component) and 14 S (small component) at 5° C to 16 and 11.5 S at 20° C (calculated m relation to assumed S-values of 23 and 16 S for the rRNA species of E. coli). We conclude that the electrophoretic mobility of RNA in polyacrylamide gels is more dependent on secondary structure than previous work had suggested and that caution is needed to interpret the relative mobility of mitochondrial RNA on gels in terms of molecular weight. In addition, our results show that judicious variation of temperature and ionic strength may allow the separation on gels of RNA mixtures that cannot be resolved under standard conditions. [ABSTRACT FROM AUTHOR]

Chemically synthesized peptidyl-tRNAPhe reacts with puromycin when bound non-enzymatirally to Escherichia coli ribosomes in the presence of poly U. Guanosine triphosphate and G factor have no influence upon the puromycin reaction, when the magnesium ion concentration is 0.01 M or lower, but stimulate the puromycin reaction when the binding of peptidyl-tRNA is carried out at higher magnesium ion concentration. In this stimulation β,γ-methylene-guanosine triphosphate cannot replace GTP, and fusidic acid inhibits the stimulation. The percentage of bound peptidyl-tRNA which reacts with puromycin in the absence of G factor and GTP decreases upon increasing the magnesium ion concentration. From all these observations one can conclude that at magnesium ion concentrations below 0.01 M, peptidyl-tRNA (such as Gly2-Phe-tRNA) binds preferentially to the donor site, but at higher magnesium ion concentrations (such as 0.025 M) the ribosomal acceptor site is preferred. Peptidyl-tRNAPhe can react with puromycin in the absence of the proper mRNA at rather high magnesium ion concentration (0.02 M and higher) but this interaction in the absence of mRNA may be not a common feature of other peptidyl-tRNAs, except tRNAMet. Phe-tRNA interacts with puromycin but the rate of this interaction is much slower than that of peptidyl-tRNAPhe such as Gly2-Phe-tRNA. [ABSTRACT FROM AUTHOR]

1. A study of sensitivity to T1 ribonuclease of the 16 S and 23 S RNAs within the 30 S and 50 S subunits and the 70 S ribosomes from Escherichia coli has been performed using sucrose gradient sedimentation. 2. The sensitivity of 23 S RNA within the 50 S subunits is greater than that of 16 S RNA within the 30 S subunits. 3. After action of T1 ribonuclease on both 30 S and 50 S subunits, under conditions which would lead to total hydrolysis of the free RNAs, the persistence of protected RNA fragments of large size is observed. 4. A much less significant degradation of 16 S and 23 S RNAs is observed, when T1 ribonuclease action is performed under the same conditions upon the 70 S ribosomes, instead of 30 and 50 S subunits; this supports the existence of regions m both RNAs involved in the association of the two subunits. [ABSTRACT FROM AUTHOR]

Electrophoretic separation in agar gel has been achieved for ribosomes from rat liver and Escherichia coli and their subunits. These particles were characterized by their mobility. RNA was extracted from the agar gel zones containing the two subunits of rat liver. It was found that the faster subunit contained only 18 S RNA with a base composition identical with that of 18 S cytoplasmic rat liver RNA. The slow subunit contained 18 S and 28 S RNA, both having the base composition of 28 S cytoplasmic RNA. The technique also provides a means of studying the labelling pattern of the ribosomes, contaminants being quantitatively removed by washing of the gel with acid. [ABSTRACT FROM AUTHOR]

A systematic study has been made of the dissociation into subunits of the ribosomes of Escherichia coli. By analytical ultracentrifugation, ribosomes were shown to be in dynamic reversible equilibrium with their subunits. This enabled a thermodynamic analysis to be made of the association-dissociation reaction and of several factors which influence it: the concentration of monovalent and divalent cations, the presence of transfer RNA and supernatant factors, the pH, and hydrostatic pressure. The apparent change in free energy for the association in 10 mM sodium phosphate pH 7.0 was -17.4 kcal/mol. Association was accompanied by the net binding of magnesium ions or the net release of monovalent cations (potassium, sodium, or Tris ions). Circular dichroism measurements of ribosomes and their isolated subunits over a range of magnesium and potassium ion concentrations indicated that a change in the conformation of each subunit accompanied association. The change occurring in the 50-S subunit was dependent on the presence of potassium ions, whereas the change in the 30-S subunit was magnesium-ion dependent. The conformational change in the 50-S subunit was critical for the association-dissociation reaction. Under ionic conditions which lead to ribosome dissociation one molecule of transfer RNA was released per 70-S ribosome. This was an independent reaction which did not influence the subunit association-dissociation equilibrium. The release of transfer RNA was accompanied by the net release of four magnesium ions, and occurred with an apparent change in the free energy of 20.8 kcal/mol. [ABSTRACT FROM AUTHOR]

1. 30-S ribosomal subparticles were hydrolysed with ribonuclease T1 or pancreatic ribonuclease in the presence of 2 M urea. The RNA · protein fragments produced were separated on 5% polyacrylamide/0.50% agarose composite gels. Fractions from the composite gels were subjected to protein analysis on 17.5% periodate-soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published. 2. Several RNA · protein fragments were obtained by this procedure, each containing equimolar amounts of a few specific ribosomal proteins. Strict criteria for specificity of the proteins in each fragment were applied. Proteins S8 and S15 were found reproducibly in a small RNA · protein fragment, with only trace amounts of the remaining ribosomal proteins. Six proteins (S7, S9, S10, S13, S14, and S19) constitute another group, and fragments containing three, four, five, or all six of these proteins were observed. Protein S20 was occasionally associated with this group, or with S6, S16 or S17, and possibly S21. The latter (including S20) were also found in association with S8 and S15. 3. The individual proteins found in specific fragments are interpreted as being close neighbours in the 30-S particle. The fragment data can be incorporated into a re-arranged version of Nomura's "assembly map". [ABSTRACT FROM AUTHOR]