An Attempt at the Identification of the Proteins Involved in the Incorporation of 5‐S RNA during 50‐S Ribosomal Subunit Assembly

Abstract

I n an attempt to determine the RNA-protein interactions involved in the biogenesis of the 50-S ribosomal subunit we have developed a simple and specific system in vitro which reflects some of the intricacies of the Escherichia coli 50-5 subunit. By studying the interaction of isolated ribosomal proteins with 5-5 and 23-S RNAs we have demonstrated the formation of a stable complex which contains 5-S RNA and 23-S RNA and which is dependent upon only two or three ribosomal proteins. The characteristics of this complex have been determined and the responsible proteins identified. The 5-5 -23-5 RNA complex is dependent upon protein L18 in conjunction with either L6 or L25. Although it would be premature to conclude that these results in vitro are directly related to the assembly map in vivo of the 50-S subunit, there is a strong correlation between our results and those obtained with various natural and artificial ribosomal subparticles.The interactions occurring between ribosomal proteins and ribosomal RNA within the intact ribosome appear to be quite complicated and involve linear as well as tridimensional structural information on the part of the RNA. Since the sequential reconstitution of the Escherichia coli 30-5 ribosomal subunit in vitro [l], there has been considerable progress made in understanding some of the RNA-protein interactions involved in both the structure and function offhe 30-S subunit [2--61; however, the larger 50-5 ribosomal subunit of E . coli which contains approximately 34 proteins and two RNA molecules [S], presents a larger problem.There are a few recent experimental reports which discuss the topography and arrangement of the 50-5 ribosomal proteins and RNA while in the complete particle. Most of these studies with E. coli have used either controlled release or partial digestion of the ribosomal proteins [7-91. Studies on the location of ribosomal RNAs within the subunit have proved equally difficult, although one laboratory has tried to localize the protein-binding sites on the 23-S RNA by partial degradation of the 50-S subunit into two fragment particles by cleavage of the 23-5 RNA, and subsequent deterDefinition. A,,, unit, 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-cm path-length cell. mination of the protein content of the isolated fragment particles [lo].Another approach to the RNA-protein problem, and the one which we have chosen, is the study of direct interaction of isolated proteins with purified RNAs. It has been shown, using a oneprotein-one-RNA system, that there are only eight 50-5 proteins which are able to bind independently and in equimolar amounts to 23-5 RNA [ll]. These very simple complexes could therefore aid in determining the precise RNA sequences involved in protein-RNA recognition. However, the functionally complete 50-5 contains 5-5 RNA as well and it would be advantageous to study the direct interactions between both RNAs and any proteins necessary for the formation of a complex containi...

A fragment of 23S RNA containing a nucleotide sequence complementary to a region of 5S RNA

“…Thus, in addition to the 5S RNA-protein interactions and 23s RNA-protein interactions hitherto reported [ 14,151, the model presented here predicts a specific interaction between SS RNA and 23s RNA.…”

A ribonuclease-resistant region of 5S RNA and its relation to the RNA binding sites of proteins L18 and L25

“…L25 was also prepared by treating ribosomes at pH 3.8 as described by Brosius (19). Fraction 1 proteins, which correspond to a 2 M LiCl-split fraction from 50S subunits,,were prepared and identified, as described earlier (2,20 Oligonucleotide sequence analyses of the RNA fragments. The 32p-labelled fragments were located in slab gels autoradiographically and the gel pieces were excised.…”

Degradation of a specific 5 S RNA – 23 S RNA — protein complex by pancreatic ribonuclease

“…Immunological analysis of the radioactive bands, performed by R. Garrett and G. Stoffler (manuscript in preparation), has demonstrated the presence of those proteins previously identified as being specifc for the 5 S RNA binding in 50 S subunits [2]. The fragments of 32P-5 S RNA which are responsible for the bands observed after pancreatic RNAase digestion of the complex, remain bound to the specific proteins during gel electrophoresis.…”

“…The formation of a specific complex containing 23 S RNA, 5 S RNA and only a few of the E. coli 50 S subunit proteins has been previously described [ 1,2]. The high degree of stability of this complex suggested that its degradation by a ribonuclease might lead to the identification of the portion of the 5 S RNA molecule which directly interacts with the other components.…”