A Specific Ribonucleoprotein Fragment from Escherichia coli 30‐S Ribosomes

Abstract

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"),

A Preliminary Three‐Dimensional Arrangement of the Proteins in the Escherichia coli 30‐S Ribosomal Sub‐Particle

“…We have shown [21] that the RNA from a specific RNA protein fragment can be isolated and "fingerprinted", and have described the region of the known 16-S RNA sequence [22,23] which is involved in the binding of proteins 197, S9, Sl9 and S10 or S13. If such experiments can be extended to an analysis of the RNA sequences present in the shoulder as well as the main peak of reproducible hydrolyses such as that shown in Fig.4 (fragments number 2 and 5, Table Z), then the regions of RNA corresponding t o proteins such as S9…”

Nucleotide Sequences of Escherichia coli 16‐S RNA Associated with Ribosomal Proteins S7, S9, S10, S14 and S19

“…ribonucleoprotein fragment containing these four proteins together with S10 [6], but since the yield of fragment from 32P-labelled ribosomes was very low, we were not able to make the analysis in very great detail. The RNA, however, clearly arose from the same general area of the 16-S RNA as the fragment described by Zimmermann et al [3].…”

Unabalanced Ribosomal Protein Synthesis in a Strain of Eascherichia coli Containing a Cloned, Truncated 16-S Ribosomal RNA Gene

“…Data for proteins S20 and S21 was not obtained in these experiments. The r-protein with the highest normalized ratio, S7 (9.17-10.68), is attached to the 3' half of the 16-S RNA molecule [25]. These data currently do not permit a logical explanation based solely on the concept of autogenous regulation.…”