An rRNA fragment and its antisense can alter decoding of genetic information

Abstract

rRNA plays a central role in protein synthesis and is intimately involved in the initiation, elongation, and termination stages of translation. However, the mode of its participation in these reactions, particularly as to the decoding of genetic information, remains elusive. In this paper, we describe a new approach that allowed us to identify an rRNA segment whose function is likely to be related to translation termination. By screening an expression library of random rRNA fragments, we identified a fragment of the Escherichia coli 23S rRNA (nucleotides 74 to 136) whose expression caused readthrough of UGA nonsense mutations in certain codon contexts in vivo. The antisense RNA fragment produced a similar effect, but in neither case was readthrough of UAA or UAG observed. Since termination at UGA in E. coli specifically requires release factor 2 (RF2), our data suggest that the fragments interfere with RF2-dependent termination.

FIG. 1

Diagram of the reporter system used for screening a random rRNA fragment library. In the reporter gene, trpA, the codon UAU (codes for Tyr) at codon position 115 (28) is replaced by UGA (see Materials and Methods). Readthrough of the UGA nonsense codon is required for growth on medium lacking tryptophan.

FIG. 2

Location of the 23S rRNA fragment isolated from the library. (Top) The secondary structure of 23S rRNA with the 74–136 segment indicated in boldface. A proposed RNA-RNA tertiary interaction (18, 19, 23) that involves the segment is indicated. (Bottom) The 74–136 fragment in detail. The nucleotides absolutely conserved among bacteria (9) are circled. The universally conserved nucleotides (24) are boxed.

FIG. 3

UGA readthrough caused by the antisense and sense fragments. “GROWTH” plates are the GM plates supplemented with Ind (see Materials and Methods). On these plates, general growth is monitored. “READTHROUGH” plates are the GM plates supplemented with a low level of Trp, to detect readthrough (see Materials and Methods). Bacterial patches growing on an Ind plate were replicated to the low-Trp plates (with and without IPTG) and to Ind plates (with and without IPTG) (see Materials and Methods). The “−” column corresponds to the plates without IPTG, and the “+” column corresponds to the plates with 1 mM IPTG. The “UAG,” “UGA,” and “UAA” columns correspond to the isogenic strains with the UAG, UGA, and UAA nonsense codons, respectively, at position 115 of trpA. Rows 1, 2, and 3 correspond to the strains transformed with pPOT1, pPOT1 with the sense fragment, and pPOT1 with the antisense fragment, respectively. The “GROWTH” plates were photographed after 24 h of incubation at 37°C. The “READTHROUGH” plates were photographed after 42 h of incubation at 37°C. (A negative of the photograph was scanned [SprintScan 35; Polaroid], and the figure was generated with the help of Adobe Photoshop 3.0 [Macintosh].)