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Journal of Bacteriology, September 2002, p. 5052-5057, Vol. 184, No. 18
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.18.5052-5057.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination

Alexey L. Arkov,,{dagger} Klas O. F. Hedenstierna, and Emanuel J. Murgola*

Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

Received 20 December 2001/ Accepted 21 June 2002

Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.

* Corresponding author. Mailing address: Molecular Genetics (Box 11), M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 792-8939. Fax: (713) 794-4295. E-mail: mannyj{at}mdanderson.org.

{dagger} Present address: Howard Hughes Medical Institute/Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016.

Journal of Bacteriology, September 2002, p. 5052-5057, Vol. 184, No. 18
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.18.5052-5057.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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