Studies on the Environment of Protein S7 within the 30‐S Subunit of Escherichia coli Ribosomes

Abstract

Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S 10, S 14 and S 19, together with RNA from sections 0'-D-E'-K-P-P'-E-A of the 16-S sequence.1. When 30-S subunits were hydrolysed with ribonuclease T, in the presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S19, together with minor amounts of S13 or S14. Oligonucleotide analysis of this fragment showed that it contained RNA from sections 0'-E' and P-A of the 16-S RNA, but that section K (from the middle of this area) was missing.2. It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S7 is the primary target of cross-linking of protein to RNA. By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits, the unbound proteins were removed, and RNA fragments containing covalently linked protein S7 were identified. It was possible to demonstrate that the site of cross-linking lies within the 0'-A region of the 26-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region.3. When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences, but which migrated as a single species into a polyacrylamide gel. Analysis of one of these complexes showed that it contained sequences from sections 0'-E' and P-A, but that section K was missing (cf: first paragraph above). This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein.

The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli

“…The sample was then loaded into a 5-cm slot of a 5 % polyacrylamide gel using the pH-6 system described previously [8], except that here again both gel and reservoir contained 0.03 "/, formaldehyde. Control experiments (minus formaldehyde) were made either with 32P-labelled or with 3H + l4C-1abelled subunits.…”

“…ethanol, to remove sarkosyl ( c j [24]). After 4 h at -20 "C, the precipitate was centrifuged off, dissolved in sample gel buffer, and applied to the twodimensional gel electrophoresis system [25], using the modified system of Roth and Nierhaus [26], with in addition N,N'-diallyltartardiamide in place of bisacrylamide [27,8]. Proteins were detected both by staining and by radioactive analysis.…”

“…We have recently published a partial localisation of the region of 16-S RNA which is cross-linked to protein S7 by ultraviolet irradiation [8], S7 being the only 30-S subunit protein which is covalently linked to RNA to any significant extent under the conditions used [13]. The next obvious development in this field is to determine whether the same type of information can be extracted from subunits in which many proteins are cross-linked simultaneously, since this is the situation which one would expect to arise from the use of most RNA-protein cross-linking procedures.…”

“…Despite this disadvantage, we have been able to make an identification of regions of the RNA to which some of the proteins are joined by this formaldehyde reaction, thereby establishing some new topographical groupings of proteins and RNA in both ribosomal subunits. Our approach has been the same as that used in the ultraviolet irradiation study on protein S7 [8], that is to say cross-linked ribosomal subunits are subjected to mild nuclease digestion, and the presence of cross-linked proteins running together with RNA fragments of known nucleotide sequence is determined. In the case of the 50-S subunit, we have made use of the well-known hydrolysis of the 23-S RNA into an 18-S and a 13-S fragment [19,20], and in the case of the 30-S subunit we have used the established hydrolysis conditions [4,5] which split the subunit into two ribonucleoprotein fragments of unequal size.…”

Crosslinking of N‐Acetyl‐phenylalanyl [s4U]tRNAPhe to Protein S10 in the Ribosomal P Site

“…It is interesting to point out that this RNA fragment is located near the Moreover, mild T1 hydrolysis of partially unfolded 3 0 4 subunits yields a ribonucleoprotein particle that contains this region of 16-S RNA, nucleotides 917-1391, as well as proteins S7, S9, S10. S13, S14 and S19 [33,34]. In addition, Traut et al…”