Mickey Miller and Dr. Allen Buskirk, Department of Chemistry and Biochemistry
tmRNA (transfer-messenger RNA) is a functional RNA that acts as both tRNA and mRNA to rescue ribosomes stalled on mRNA lacking a stop codon or on nascent peptide sequences in eubacteria (1). When a ribosome stalls, the tRNA-like structure of tmRNA enters the ribosome and adds an alanine to the growing peptide. The ribosome then releases the original mRNA and translation continues with tmRNA as the template. The tmRNA template codes for a 10 amino acid tag sequence followed by a stop codon. With a stop codon, the ribosome is able to dissociate and be recycled. Meanwhile, the tagged peptide is released and degraded by proteases (2).
The trans-translation model with tmRNA describes the synthesis of a single polypeptide from two RNA templates. How does the ribosome know where to resume translation on tmRNA? Model studies suggest that the single-stranded codon in the −1 position (the −1 triplet) is responsible for proper recognition of the correct reading frame (3). These studies show that solvent molecules interact with the −1 triplet to mimic the A-form RNA duplex observed in codon:anticodon duplexes. The A-form RNA duplex is the native conformation of double-stranded RNA just as the B-form duplex is the native conformation of double-stranded DNA. Analysis of models of the 64 possible −1 triplets led to the formulation of rules governing the identity of the triplet. These rules indicate that the pyrimidine bases U and C cannot precede the purine base G (or A when A is in the anti conformation), and A cannot precede G when A is in the syn conformation. Otherwise, an energetically unfavorable condition compromises the A form conformation of the −1 triplet due to the loss of hydrogen and ionic bonds. As a result, these rules prohibit the use of 18 of the 64 possible −1 triplets in tmRNA.
In this study, we created tmRNA mutants containing each of the 64 possible −1 triplet identities. By studying these mutants in vivo, we will be able to determine the effect of the −1 triplet on the function of tmRNA. The results of this study will also determine the validity of the rules mentioned above that are proposed to govern the identity of the −1 triplet in tmRNA.
The KanR selection assay (4) is a powerful tool for determining activity of tmRNA mutants. Active tmRNA mutants with the tag sequence for the last 14 amino acids of KanR rescue ribosomes stalled on truncated kanR mRNA and generate full-length KanR as a resistance to kanamycin. With this assay, we have been able to characterize the activity of all of the 64 possible −1 triplet mutants. Fully functional mutants resulted in the growth of bacterial colonies on kanamycin plates equal to, or nearly equal to the number of colonies found on control plates. Nonfunctional mutants, on the other hand, resulted in the growth of no colonies on kanamycin plates. The results of these assays have allowed us to determine the effect of the −1 triplet on activity of tmRNA. The results from the assays show that many of the −1 triplets are present in active tmRNA.
Because the ribosome recognizes the A-form codon:anticodon RNA duplex, the −1 triplet must somehow mimic the A-form duplex to allow the ribosome to resume translation on the tmRNA template. Of the 18 predicted forbidden triplets, we have found that at least 10 result in functional tmRNA mutants. Although the mutant −1 triplets do not follow the hypothesized rules for the −1 triplet, these 10 functional tmRNA mutants must be able to mimic the A-form complex at the −1 triplet. These results show that the hypothesized rules cannot predict functional −1 triplet mutants as they are currently presented.
Although we expected to characterize the −1 triplet mutants as either functional or nonfunctional in our assays, a number of mutants showed weak survival (<60%). Other studies have shown that base mutations in the −1 triplet position can lead to frameshifting in the tmRNA template (5). Future testing of these −1 triplet mutants will show whether frameshifting is a possible explanation for this weak survival or total lack of function.
The hypothesized rules predicted to govern functional −1 triplet tmRNA mutants are inadequate at best. We have shown that a number of predicted forbidden triplets result in functional tmRNA. Our results appear to show that purines are favored over pyrimidines at the first position of the −1 triplet. Further testing will hopefully introduce more information that will help us better understand the role of the −1 triplet in tmRNA.
Difficulties in cloning the final ten −1 triplet mutants slowed our progress in this project. Until we were able to produce the DNA that would give us the desired −1 triplet tmRNA mutant, we were unable to carry out the assays required to test these remaining mutants. We also had to adjust the stringency of kanamycin in our assays to try to interpret our results from a preliminary experiment. With a higher stringency, we have obtained what we believe to be more meaningful data. All difficulties that we have experienced up to this point have been resolved.
I presented my research with the −1 triplet at the 20th Annual Spring Research Conference in March 2006. Since then, we have learned much more. We will be submitting this research by the end of July to be published in either RNA or NAR (Nucleic Acid Research).
- Hayes, C. S., Bose, B., and Sauer, R. T. (2002) J. Biol. Chem. 277, 33825–33832
- Withey, J. H., and Friedman, D. I. (2003) Annu. Rev. Microbiol. 57, 101–123
- Lim, V. I., and Garber, M. B. (2005) J. Mol. Biol. 346, 395–398
- Tanner, D. R., Dewey, J. D., Miller, M. R., and Buskirk, A. R. J. Biol. Chem. (In press).
- Lee, S., Ishii, M., Tadaki, T., Muto, A., and Himeno, H. (2001) RNA 7, 999–1012