Peter Fellner

RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes.

Abstract Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the S 16 S 17 mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of S 16 S 17 was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T1 or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and S 16 S 17 as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA.

The determination of the primary structure of the 16S ribosomal RNA of Escherichia coli: (2) Nucleotide sequences of products from partial enzymatic hydrolysis

In the p reced ing paper we have reported the sequences of the ol igonucleotides ar is ing upon complete hydrolys is of the 16S r ibosomal RNA wi th T 1 and pancrea t ic r ibonucleases . In this art icle we describe the progress that has been made in a r r ang ing these ol igonucleotides in a single defined sequence. In the absence of other easily real isable possibil i t ies, we have adopted the general approach of cleaving the 16S RNA into fragments of various sizes, by par t ia l hydrolysis wi th nucleases carr ied out unde r a variety of condit ions. The fragments have been separated by gel e lectrophoresis procedures , and characterised by fur ther enzymat ic digestions, wi th reference to the ol igonucleot ide catalogues reported in the accompany ing paper.

Oligonucleotide mapping of picornavirus RNAs by two-dimensional electrophoresis.

Abstract Considerable differences have been found in two-dimensional polyacrylamide gel electrophoresis fingerprints of complete ribonuclease T 1 digestion products of the RNAs of representative members of the entero-, cardio-, and foot-and-mouth disease virus subgroups of the picornavirus family. Individual members of the different subgroups, serotypes of a virus, and even subtypes within a serotype can be distinguished by the use of this technique. The method has also facilitated the identification of homopolymeric regions within the different picornavirus genomes, and the presence of a poly(C) tract in the cardio- and foot-and-mouth disease virus subgroups has been confirmed. A poly(A)-rich tract of approx 40–100 nucleotides has been detected in all the picornaviruses studied. Oligonucleotide fragments possibly specific to the enterovirus subgroup were also detected and partially characterised.

GENERAL ORGANIZATION AND STRUCTURE OF THE PICORNAVIRUS GENOME

The picornavirus group numbers over 200 distinct serotypes, isolated from a wide variety of human and animal sources, and it is likely that many others exist in nature (1), as Vet unrecognised. Various sub-classifications of this large range of viruses have been proposed (2, 3). The most recent, and probably the most generally satisfactory classification (4, 5) arranges the picornaviruses into five genera (a) Cardioviruses, including encephalomyocarditis (EMG) and mengoviruses; (b) Human Rhinoviruses; (c) Equine Rhinoviruses; (d) Foot-and-Mouth Disease viruses; (e) Enteroviruses, including polio, Coxsackie viruses etc. The members of these different genera display differences in virus particle stability in the pH range 3–7, and in their particle density in caesium chloride, as well as in RNA base composition (see chapters 1 and 3).