1. 30-5 ribosomal subparticles were hydrolysed with ribonuclease TI or pancreatic ribonuclease in the presence of 2 M urea. The RNA * protein fragments produced were separated on 501, polyacrylamide/0.5 ,Ilo agarose composite gels. Fractions from the composite gels were subjected to protein analysis on 17.5O/, 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 58 and 515 were found reproducibly in a small RNA * protein fragment, with only trace amounts of the remaining ribosomal proteins. Six proteins (57, S9, 510, 513, 514, 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 56, 516 or 517, and possibly 521. The latter (including 520) were also found in association with S8 and 515.3. 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". I n our last paper [l] we described a method for the analysis of ribonucleoprotein (RNA * protein) fragments, which we obtained by mild nuclease digestion of Escherichia coli 30-5 ribosomes. The underlying objective of the work was that a precise analysis of small specific ribosomal fragments provides a direct method for examining the arrangement of proteins and RNA within the particle. I n this paper, we apply the technique to a number of RNA .protein fragments, produced under a variety of different hydrolysis conditions, and discuss the results in relation to Nomura's "assembly map" [2,31. MATERIALS AND METHODS Preparation of Ribosomes30-5 ribosomal subparticles were prepared from E. coli MRE 600 (obtained from MRE, Porton, U.K.) as described before [1,4], with the following modificaAbbreviations. RNA * protein, ribonucleoprotein; sarkosyl, N-lauryl sarcosine.Enzymes. T, ribonuclease (EC 2.7.7.26); pancreatic ribonuclease (EC 2.7.7.16).Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-em path length cell.tions. After grinding with alumina [a], the cell paste was extracted with 10 mM MgC1, rather than 0.1 mM, to maintain the ribosomes as 70-5 particles. These 70-5 particles were washed by spinning through 0.5 M NH4Cl, 10 mM MgCl,, 10 mM Tris-HC1 pH 7.6 (cf. [5]), and were then dissociated into subparticles by resuspending in 50mM KC1, 0.3 mM MgCl,, 10mM Tris-HC1 p H 7.6. The subparticles were separated in a zonal rotor as before [I], and the 30-5 ribosomes precipitated with ethanol. The precipitate was dissolved in 0.3 mM magnesium acetate, 10 mM Tris-HC1 pH 7.6, and dialysed against this buffer, or against 1 mM magnesium...

1. 30-S ribosomal subparticles from Escherichia mli were hydrolysed with ribonuclease TI, pancreatic ribonuclease or micrococcal nuclease in the presence of 2 M urea, and various concentrations of magnesium and ethanol. The RNA -protein fragments produced were separated on 50l0 polyacrylamidelagarose composite gels, and fractions from these gels were subjected to protein analysis on i7.5O/, periodate-soluble polyacrylamide gels run in the detergent sarkosyl, using the technique already published.2. A wide range of RNA * protein fragments was obtained by this procedure, each containing a few specific ribosomal proteins. The strict criteria already published for determining the specificity of the proteins in each fragment were applied. The RNA -protein fragments divide into two distinct groups, those containing some or all of proteins 57, S9, SiO, 513, Si4 and Si9, and those containing some or all of proteins S4, 55, S6, S8, Sii, Sl5, S16(i7), 518 and S20. Proteins S1, S2, 53, Si2 and 521 were not found in specific fragments. 3.The individual proteins found together in specific RNA -protein fragments are interpreted as being close neighbours in the 30-S particle. The range of fragments observed is sufficient to enable the data to be combined with Nomura's "assembly map)' and data from protein crosslinking experiments, into a preliminary three-dimensional arrangement of the proteins.I n previous papers we have described a method for the analysis of ribonucleoprotein (RNA -protein) fragments from Escherichia coli ribosomes [i], and have used this method to characterize a series of specific fragments from the 30-5 particle [2]. I n this paper we present a further series of fragments, obtained by mild nuclease digestion of the 30-S ribosome with ribonuclease TI, micrococcal nuclease or pancreatic ribonuclease. These two series of fragments account for 16 out of the 21 ribosomal proteins, and the data have been combined with the assembly map Definition. Azao unit is the quantity of material contained in I ml of a solution which has an absorbance of I at 260 nxn, when measured in a 1-cm path-length cell. MATERIALS AND METHODS Preparation of RibosomesRadioactive and non-radioactive 30-5 ribosomal sub-particles from E . coli MRE 600 (obtained from MRE, Porton, U.K.) were prepared exactly as described previously [2], except that the isolated subparticles were kept stored at -20 "C in 10 mM Tris-HC1 pH 7.8, 0.3 mM magnesium acetate, Containing 10-20°/0 ethanol. The ethanol was only dialysed away immediately before use in hydrolysis reactions. Separation and Analysis of RNA .Protein FragmentsRadioactive 30-S ribosomes were hydrolysed with ribonuclease TI, pancreatic ribonuclease, or micrococcal nuclease (all from Sigma) for 4.5 h at room temperature. Reaction mixtures contained 8-10 ABso units of ribosomes in 0.2-0.4 ml. The hydrolysates were separated and analysed by electrophoresis on 501, polyacrylamide/0.5 agarose composite gel slabs

1. 32P-labelled ribosomal sub-particles were hydrolysed with ribonuclease T,, under conditions which have previously been shown to yield a specific fragment of RNA . protein ("band III"), containing only four of the 21 30-S ribosomal proteins. The hydrolysate was fractionated on a polyacrylamide gel using the methods already published, and gel fractions containing the specific RNA . protein fragment were subjected to electrophoresis on a second polyacrylamide gel, in the presence of sodium dodecylsulphate to dissociate RNA and protein. Two bands of [32P]RNA were reproducibly observed, containing about 320 and 360 nucleotides, respectively.2. Two-dimensional fingerprints of ribonuclease T, hydrolysates from both these RNA species showed that the two fragments were almost identical. Oligonucleotides from the fingerprints were digested with pancreatic ribonuclease, and the secondary digestion products analysed and compared with the known sequence of 16-S ribosomal RNA. Several characteristic oligonucleotides from regions D, E and P of the RNA were identified unambiguously. Other oligonucleotides were found which could have arisen from more than one part of the 16-8 RNA, but in each case one of the possibilities was in regions D, E, E' or P.3. It was concluded that the band I11 proteins are associated with these contiguous regions of the 16-S RNA, i.e. near to its 3'-end.The topography of the 30-5 ribosomal subparticle of Escherichia coli is a subject which is currently attracting a great deal of attention, both with regard to the interactions between individual proteins and their binding to the ribosomal RNA. The problem of elucidating the binding sites or rather the areas of ribosomal RNA to which individual proteins are bound has been approached in different ways. Definition. An A,,, unit is the quantity of material contained in 1 ml of a solution which has an absorbance of 1 a t 2GO nm, when measured in a 1-cm path-length cell.[2] have investigated the interaction of individual proteins with specific large fragments of 16-S RNA, and have shown that proteins S4, S8, S15, S20 and possibly 513 bind to the 5'-terminal half of the RNA, whereas protein S7 interacts with the 3'-terminal half of the molecule. Our approach has been to isolate and analyse specific ribonucleoprotein (RNA . protein) fragments of the 30-S particle [3,4], in the hope that we would not only be able to discover which proteins are adjacent to each other in the ribosome, but also that we could determine the general area of the 16-S RNA responsible for binding some groups of proteins. We were interested in particular in those proteins which cannot be studied by direct binding to RNA under reconstitution conditions [2].I n this paper, we describe our studies on the RNA moiety of the RNA . protein fragment "band III".This fragment was shown earlier [3,4] to contain proteins S7, S9, S13, occasionally Sl0, and either S l 4 or S19. By establishing some unique nucleotide sequences in the RNA of this fragment ("RNA III"),

1. 305 ribosomal subunits from Escherichia coli were hydrolyzed with ribonuclease T,, and the ribonucleoprotein fragments produced were fractionated on a 5 ,Ilo polyacrylamide/0.5 ,Ilo agarose composite gel. Two main breakdown products were observed corresponding in size to about 20°/, and 60°/, of the intact 305 particle.2. Each fraction from the 501, composite gel was subjected to protein analysis on a periodatesoluble 15 ,Ilo polyacrylamide gel, run in the detergent Sarkosyl. The smaller ribonucleotprotein fragment was found to contain only four of the 30s ribosomal proteins, and these were present in equimolar amounts. The larger fragment had a complex protein composition ; it was deficient in the four proteins contained in the smaller fragment, and was considerably enriched in the largest 30s protein. 3.The results show a striking correlation with Nomura's "assembly map" of the 30s particle.I n a recent paper [l], we described a method for the isolation and analysis of ribonucleoprotein fragments from the smaller subparticle of Escherichia coli ribosomes. We suggested that analysis of such fragments, obtained by controlled digestion with ribonuclease, offered a new approach to the problem of "mapping" the proteins within the ribosome. The method is essentially a two-step polyacrylamide gel electrophoresis; the first step separates the RNA * protein fragments, and the second separates the individual proteins within the fragments. I n this paper, we describe various refinements to our polyacrylamide-gel techniques, which now make it possible to define the specific protein composition of an RNA protein fragment with precision. Cleavage of 50s ribosomal particles into specific fragments has recently been reported by other workers [2]. Definition. A,,, unit, the quantity of material contained i n 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-cm path length cell. MATERIALS AND METHODS Preparation

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