Richard L. Gourse

Fine structure of ribosomal RNA. III. Location of evolutionarily conserved regions within ribosomal DNA.

Abstract In order to define functional regions within ribosomal RNA, we have identified areas of the molecule which have been conserved during evolution. Our previous studies showed that there is evolutionary conservation between the rRNAs of different eukaryotes and that the sequences conserved between distantly related species are a subset of those conserved between closely related species. In the present work, we have employed DNA-DNA and DNA-RNA hybridization techniques to localize these conserved regions to mapped restriction fragments 50 to 300 base-pairs in length within cloned Xenopus laevis ribosomal DNA. Our experiments have detected evolutionary conservation only within the coding regions, suggesting that if there is any conservation within the spacers, these sequences must be very short. Regions of conservation can be classified either by evolutionary distance or by the extent of conservation between two species. Three regions, including one near the 3 end of 18 S and two near the 3 end of 28 S rRNA are conserved over great evolutionary distance, that is between Escherichia coli and X. laevis. In addition, several fragments in the central portions of the 188 and 28 S rRNAs are exceptional in the extent of their conservation between yeast and Xenopus. We have been able to correlate the regions we have defined as conserved with certain structural or functional roles, such as initiation of translation, possible interaction with transfer RNA, rRNA methylation, and the site where intervening sequences interrupt some eukaryotic rRNAs. As a result, these studies serve to define relatively short (less than 300 base-pairs) segments within the almost 11,000 base X. laevis rDNA repeat unit which are worthy of further investigation.

Interaction of ribosomal proteins S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA from Escherichia coli☆

The co-operative interaction of 30 S ribosomal subunit proteins S6, S8, S15 and S18 with 16 S ribosomal RNA from Escherichia coli was studied by (1) determining how the binding of each protein is influenced by the others and (2) characterizing a series of protein-rRNA fragment complexes. Whereas S8 and S15 are known to associate independently with the 16 S rRNA, binding of S18 depended upon S8 and S15, and binding of S6 was found to require S8, S15 and S18. Ribonucleoprotein (RNP) fragments were derived from the S8-, S8/S15- and S6/S8/S15/S18-16 S rRNA complexes by partial RNase hydrolysis and isolated by electrophoresis through Mg2+-containing polyacrylamide gels or by centrifugation through sucrose gradients. Identification of the proteins associated with each RNP by gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated the presence of S8, S8 + S15 and S6 + S8 + S15 + S18 in the corresponding fragment complexes. Analysis of the rRNA components of the RNP particles confirmed that S8 was bound to nucleotides 583 to 605 and 624 to 653, and that S8 and S15 were associated with nucleotides 583 to 605, 624 to 672 and 733 to 757. Proteins S6, S8, S15 and S18 were shown to protect nucleotides 563 to 605, 624 to 680, 702 to 770, 818 to 839 and 844 to 891, which span the entire central domain of the 16 S rRNA molecule (nucleotides 560 to 890). The binding site for each protein contains helical elements as well as single-stranded internal loops ranging in size from a single bulged nucleotide to 20 bases. Three terminal loops and one stem-loop structure within the central domain of the 16 S rRNA were not protected in the four-protein complex. Interestingly, bases within or very close to these unprotected regions have been shown to be accessible to chemical and enzymatic probes in 30 S subunits but not in 70 S ribosomes. Furthermore, nucleotides adjacent to one of the unprotected loops have been cross-linked to a region near the 3 end of 16 S rRNA. Our observations and those of others suggest that the bases in this domain that are not sequestered by interactions with S6, S8, S15 or S18 play a role involved in subunit association or in tertiary interactions between portions of the rRNA chain that are distant from one-another in the primary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Site-directed mutagenesis of ribosomal RNA: Analysis of ribosomal RNA deletion mutants using maxicells☆☆☆

Abstract Until now, a genetic approach to the study of ribosomal RNA (rRNA) has been limited by the absence of mutants coding altered rRNA. In the accompanying paper by Gourse et al. we have described the construction and characterisation of plasmids coding mutant rRNAs. The mutant plasmids each contain a small deletion at one of seven chosen sites in the rRNA coding regions. In order to study the effects of these mutations we have modified the maxicell system of Sancar et al. (1979). We have obtained expression of plasmid-coded rRNA in the complete absence of host-coded rRNA synthesis. Optimal yield and specificity for synthesis of plasmid-coded rRNA were obtained using maxicells generated by use of low ultraviolet light fluences. Under these conditions, maxicells synthesized many proteins with no apparent specificity for expression of only the plasmid-coded polypeptides, despite complete specificity for expression of only the plasmid-coded rRNA. In maxicells containing the wild-type (i.e. unaltered) plasmid, rRNA transcripts were fully processed and assembled into ribosomal subunits. Both 30 S and 50 S subunits assembled in maxicells were matured sufficiently to form 70 S ribosomes. However, small deletions at positions 704 and 1504 in 16 S rRNA abolished the maturation of the mutant precursor 16 S (17 S) rRNAs and concurrently prevented completion of 30 S subunit assembly. Even single-base deletions at two other sites (positions 615 and 1384/1385 in 16 S rRNA) severely retarded both precursor rRNA maturation and 30 S subunit assembly. Mutations at two sites in 23 S rRNA (positions 607 and 1985) had no apparent effect on the processing of the mutant precursor 23 S rRNAs, but partially reduced the production of mature 50 S subunits. Small deletions at position 365 in 23 S rRNA prevented its assembly into 50 S subunits with the capacity to form 70 S ribosomes, although processing of the mutant precursor 23 S rRNA was not prevented.

Site-directed mutagenesis of ribosomal RNA: Construction and characterization of deletion mutants

Abstract We have used a genetic approach to the study of ribosomal RNA structure and function by using in vitro, site-directed mutagenesis techniques on a plasmid carrying the rrnB rRNA operon of Escherichia coli. By limited exonuclease digestion from chosen restriction sites in the parent plasmid, a series of mutant E. coli strains was constructed, each with a deletion at one of seven positions corresponding to bases 614, 704, 1384 and 1504 in 16 S rRNA and 365, 607 and 1984 in 23 S rRNA. We have selected for study plasmids containing small deletions of 1 to 30 bases in length in order to correlate distinct regions of the rRNA molecule with rRNA processing, its assembly into ribosomal subunits, and its function in ribosomes. The deletions were characterized by restriction enzyme analysis and DNA sequencing, and the mutant rRNAs were shown to be transcribed in vivo. The growth rates of strains that carry the altered plasmids, in addition to their normal complement of seven wild-type rRNA cistrons, are in many cases severely retarded. Some strains carrying plasmids with only a single base deletion have doubling times as much as twice that of the strain carrying the unaltered plasmid. In the case of one mutant, there is a spontaneous deletion at a second site that partially suppresses the effects of an initial single base mutation in 23 S RNA. The methodology described here for the construction and propagation of clones containing rDNA mutations provides an alternative approach to the study of rRNA structure and function. The accompanying paper by Stark et al. describes the analysis of the altered transcripts, their processing, assembly into subunits, and incorporation into ribosomes by a method that allows their detection separately from the host-encoded rRNA.

Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly.

Using a multicopy plasmid encoding the Escherichia coli rrnB ribosomal RNA operon and the techniques of in vitro site-directed mutagenesis, we have introduced several small alterations into the central domain of 16 S rRNA, which encompasses nucleotides 560 to 890. Four of the rRNAs studied contained deletions and one contained an insertion. The altered small ribosomal subunit rRNAs were used to investigate relationships among 16 S rRNA processing, protein-16 S rRNA interactions and assembly of the 30 S ribosomal subunit. Analysis of plasmid-coded transcripts from maxicells revealed that products from wild-type 16 S rRNA genes were fully processed and assembled into mature 30 S subunits. Under the same conditions, the processing and assembly of transcripts derived from the mutant plasmids were severely impaired. In some instances, the mutations completely blocked both processes, while in other cases rRNA maturation and ribosome assembly were retarded, but not eliminated completely. In all cases, the mutations led to the accumulation of the 17 S precursor to 16 S rRNA. The mutant 17 S rRNAs were purified and incubated with various combinations of E. coli ribosomal proteins S6, S8, S15 and S18, which are known to bind to the central domain of 16 S rRNA. Ribonuclease digestion of the resulting protein-17 S rRNA complexes and fractionation of the products permitted detection of three distinct protein-RNA fragment complexes which contained S8, S8 + S15, or S6 + S8 + S15 + S18. Whereas wild-type 17 S rRNA was able to form all three of these complexes, deletion of nucleotides 693 to 721 or 822 to 874 abolished the interaction of S6 and S18, and removal of nucleotides 659 to 718 prevented the binding of S6, S15 and S18. In contrast, elimination of residue 614, or the presence of a 16-base insertion between nucleotides 614 and 615, had no significant effect on the binding of any of the four proteins tested. Together, our results demonstrate that 16 S rRNA maturation and 30 S subunit assembly are tightly coupled, and show that, in at least some cases, defects in these processes can be correlated with the inability of particular ribosomal proteins to associate with altered rRNA molecules. Moreover, we have confirmed the essentiality of certain rRNA sequences for the formation and/or stabilization of these protein-rRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Regions of DNA involved in the stringent control of plasmid-encoded rRNA in vivo

We have examined the transcription of two plasmid-encoded, stable RNAs; a shortened 16S ribosomal RNA and the spacer transfer RNA2Glu from the Escherichia coli rrnB operon. Plasmid deletions were constructed in vitro, in order to examine the DNA regions required for stringent control of rRNA expression in vivo during amino acid starvation. We find that rRNA synthesized from plasmids does exhibit a relA-dependent, stringent response. The DNA sequences required for this regulation do not extend beyond 20 bases downstream of the P1 transcription initiation site. Deletion of P2, the second of the two tandem rRNA promoters, does not weaken the stringent control of transcripts from P1. These results demonstrate that pause sites for RNA polymerase identified in vitro do not play a significant role in the stringent control of rRNA synthesis in vivo and imply that stringent regulation takes place at the level of initiation, rather than elongation, of transcription. Surprisingly, we find that the presence of extra intact rrnB operons (carried by a multicopy plasmid) reduces the magnitude of the stringent response.

A mutation in an Escherichia coli ribosomal RNA operon that blocks the production of precursor 23 S ribosomal RNA by RNase III in vivo and in vitro

We have isolated on a multicopy plasmid a mutant rrnB ribosomal RNA operon containing a 130 base-pair deletion immediately preceding the 23 S rRNA gene. The deletion shortens by just three base-pairs the 26 base-pair complementarity of the sequences that flank the 23 S rRNA gene, and which normally form an RNase III cleavage site in the rrnB primary transcript. Both in vivo and in vitro, cleavage at the altered RNase III site was almost completely abolished by the mutation. Our results therefore indicate that even a small perturbation of the double-stranded region normally recognized by RNase III strongly inhibits the action of the enzyme.