Evidence for functional interaction between domains II and V of 23S ribosomal RNA from an erythromycin-resistant mutant.

Abstract

A mutation affording low levels of erythromycin resistance has been obtained by in vitro hydroxylamine mutagenesis of a cloned ribosomal RNA operon from Escherichia coli. The site of the mutational event responsible for antibiotic resistance was localized to the gene region encoding domain II of23S rRNA by replacement of restriction fragments in the wild-type plasmid by corresponding fragments from the mutant plasmid. DNA sequencing showed that positions 1219-1230 of the 23S rRNA gene are deleted in the mutant. Since all previously characterized rRNA mutations conferring resistance to erythromycin show changes exclusively in domain V, our present findings provide direct evidence for functional interaction between domains II and V of 23S rRNA.Efforts to understand the role ofrRNA in translation have led to the identification ofseveral putative functional sites in 16S-and 23S-like rRNAs (reviewed in ref. 1). Our present knowledge of the structural organization of these sites is limited to a description of their secondary structures and, in some cases, their locations relative to the electron microscopic models for the ribosomal subunits (reviewed in ref.2). Deeper insight into the mechanisms of rRNA function would undoubtedly result from information relevant to the three-dimensional arrangement of these sites.The secondary structure models for the rRNAs suggest that they are organized into discrete structural domains (1). The strong phylogenetic conservation ofthese domains raises the question as to whether this has some important functional basis, as has been established for many proteins. Specifically, is it possible that there is functional interaction between rRNA domains during the course of protein synthesis? One of the most direct approaches, chemical crosslinking, has provided evidence for close proximity between specific regions of different rRNA domains (reviewed in ref.3).Genetic approaches, which are potentially among the most powerful in exploring the functional and structural organization of rRNA, have yet to be extensively exploited in this context. To a great extent, this is because of the difficulty of performing genetic analysis on an essential gene that is present in seven copies in the genome ofEscherichia coli, the reference organism for study of ribosomes. This problem has been alleviated by using rRNA operons that have been cloned in high-copy-number plasmids. This approach has permitted isolation of rRNA mutants from E. coli that show observable phenotypic changes (4), including antibiotic resistance (5).In this paper we describe an erythromycin-resistant mutant, obtained by random in vitro mutagenesis of a cloned rRNA operon. The site of mutation lies in domain II of 235 rRNA, remote from all previously identified erythromycinresistant rRNA mutations, which have been localized to a small region in domain V (6, 7). We take this to be direct evidence for functional interaction between two different domains in a rRNA. MATERIALS AND METHODSCell Strains and Medium. E. coli strain DH1 [F...

Functional interactions within 23S rRNA involving the peptidyltransferase center

“…1), orienting positions 2032 and 2058 in the midst of domain II. A functional connection between domains II and V based on the A12 mutation that confers erythromycin resistance has previously been reported (13). Expression of A12 23S RNA from the rnB promoters slows cell growth in the absence of erythromycin (Table 1), probably because the deletion has a side effect on 50S subunit assembly (12).…”

“…2b. The formation of the 2058G mutation and A12 (a 12-bp deletion of nucleotides 1219 to 1230) has been previously described (13,36). 23S…”

“…1) Table 2, the rrnB promoters have been replaced by the lambdapL promoter (12,19). Plasmid pSK41 contains the 4.3-kb XbaI-BamHI 3' portion of rrnB (13). rates of wild-type, 2032C, or 2032U mutants at permissive levels of clindamycin (10 mg/liter).…”

Mutations in the Intersubunit Bridge Regions of 23 S rRNA

“…Bacterial Strains and Plasmids-The rRNA mutations were initially constructed in plasmid pLK35, in which the rrnB operon is transcribed from the P L promoter (13). Strain pop2136 carries the temperature-sensitive cI repressor, and in this strain, transcription of plasmid-encoded rRNA is repressed at 30°C but can be induced by a temperature shift to 42°C (14).…”

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…Additionally, other alterations have been described in Enterobacteriaceae, such as those affecting positions A745, A752, U754, G2057, A2032, A2062, A2503, U2609, C2610 or C2611, which are able to affect the activity of macrolides either by increasing the levels of resistance, or resulting in enhanced activity. Moreover, an association has been described between a specific 23S rRNA deletion (D1219-1230) and the development of erythromycin resistance (Douthwaite et al, 1985;Krokidis et al, 2014;Novotny et al, 2004;V azquez-Laslop et al, 2008V azquez-Laslop et al, , 2010V azquez-Laslop et al, , 2011Vannuffel et al, 1992, Weisblum, 1995 (Table 3). Alterations present in domain II of the 23S rRNA affect specific macrolides like tylosin and ketolide antibiotics but not erythromycin or azithromycin.…”