Polyadenylation helps regulate functional tRNA levels in Escherichia coli

Bijoy K. Mohanty 0 Valerie F. Maples 0 Sidney R. Kushner 0 0 Department of Genetics, University of Georgia , Athens, GA 30602, USA Here we demonstrate a new regulatory mechanism for tRNA processing in Escherichia coli whereby RNase T and RNase PH, the two primary 30 ! 50 exonucleases involved in the final step of 30-end maturation, compete with poly(A) polymerase I (PAP I) for tRNA precursors in wild-type cells. In the absence of both RNase T and RNase PH, there is a >30-fold increase of PAP I-dependent poly(A) tails that are 10 nt in length coupled with a 2.3- to 4.2-fold decrease in the level of aminoacylated tRNAs and a >2-fold decrease in growth rate. Only 7 out of 86 tRNAs are not regulated by this mechanism and are also not substrates for RNase T, RNase PH or PAP I. Surprisingly, neither PNPase nor RNase II has any effect on tRNA poly(A) tail length. Our data suggest that the polyadenylation of tRNAs by PAP I likely proceeds in a distributive fashion unlike what is observed with mRNAs. - In Escherichia coli, diverse processing pathways employing RNase E, RNase P, polynucleotide phosphorylase (PNPase) and RNase II are used to generate pre-tRNA species from primary transcripts (15). Subsequently, the pre-tRNAs undergo additional maturation at either both their 30- and 50-ends or at their 30-ends only (310) to generate functional tRNAs. It has been shown that RNase T, RNase PH, RNase D, RNase BN are the four 30 ! 50 exoribonucleases that primarily participate in 30-end maturation of tRNAs and many other stable RNAs (6,7,9), although RNase BN has endonucleolytic activity in vivo and is also called RNase Z (1113). Interestingly, E. coli can use some incompletely processed stable RNAs (rRNAs) without significant effects on their growth phenotype (8,1416), but tRNAs are an exception. All tRNAs, which account for the major fraction of stable RNAs in the cell (17), must be completely processed at their 30-termini before they can be aminoacylated and used in protein synthesis (18). Although the 30-ends of some tRNA precursors are probably matured by a preferred set of exonucleases (3,5,9), it is thought that the majority of the 30-end tRNA maturation is carried out by RNase T and RNase PH with only minor contributions by RNase D and RNase BN (9). Taken together these results present a comprehensive overview of the processing of primary tRNA transcripts into functional species. However, the observation of polyadenylated tRNAs in the absence of both RNase T and RNase PH (16,19) was rather unexpected, particularly since polyadenylation in E. coli by poly(A) polymerase I (PAP I) has been almost exclusively characterized in relation to mRNAs (2023). Furthermore, sequencing analysis of hisR, cysT and leuX transcripts has shown that a significant fraction ( 2033%) of the transcripts have short poly(A) tails in a RNase PH single mutant (4,5). Interestingly, the fraction of normal pre-tRNAs (not defective based on nucleotide sequence) with poly(A) tails was considerably higher than previously observed for other transcripts (mRNAs and rRNA) in E. coli (24,25). Although no poly(A) tails have been detected on mature tRNAs or 5S rRNA in wild-type E. coli, Li et al. (26) proposed a model in which the primary function of polyadenylation was to identify and present defective tRNA processing intermediates for recycling through degradation pathway(s) that are part of a general quality control process. The evidence for poly(A)-dependent degradation of a mutant tRNATrp (26) and various mRNAs (22,24,27) provided support for this hypothesis, but it did not explain the presence of polyadenylated pre-tRNA transcripts in the rph-1 mutant that were not defective (4,5). Similarly, it has been suggested that the shorter poly(A) tails observed on many stable RNA precursors resulted from degradation of longer poly(A) tails by exoribonucleases such as polynucleotide phosphorylase (PNPase), RNase II or RNase R (19). However, inactivating either PNPase or RNase II did not change the length of poly(A) tails associated with leuX transcripts (5). Taken together, these data suggested a potentially more significant role for the observed polyadenylation of pre-tRNAs in E. coli. Here, we present a detailed analysis of 50 out of 86 tRNA primary transcripts along with 5S rRNA that demonstrates a second function for RNase T and RNase PH beyond their role in tRNA 30-end maturation. Specifically, both enzymes reduce or block the addition of short poly(A) tails by PAP I on tRNAs, with RNase T being more effective than RNase PH. Furthermore, polyadenylation induces rapid degradation of 5S rRNA precursors but only a small fraction of tRNA precursors. Rather the majority of polyadenylated precursors are slowly converted into mature species, most likely by RNase D and/or RNase BN. The data are consistent with the 2- to 4-fold drop in the pool of aminoacylated tRNA species along with a concomitant >2-fold increase in cell generation time in an Drnt rph-1 double mutant. In contrast, charged tRNA levels and growth rate improved significantly in a DpcnB Drnt rph-1 triple mutant. Furthermore, a small number of tRNAs (7/86) are resistant to polyadenylation even in the absence of both RNase T and RNase PH. Of particular interest is the fact that PAP I apparently acts on tRNAs substrates in a distributive manner compared to a more processive mechanism for mRNAs. MATERIALS AND METHODS Bacterial strains and plasmids The E. coli strains used in this study were all derived from MG1693 (rph-1 thyA715) (E. coli Genetic Stock Center, Yale University). This strain contains no RNase PH activity and has reduced expression of pyrE because of a single nucleotide frameshift in the rph gene (28). A rph+ derivative was constructed by transducing a P1 lysate grown on E. coli C600 into MG1693 and selecting for faster growing isolates on minimal medium. Several independent transductants were sequenced to confirm the presence of the wild-type rph coding sequence. One such isolate was designated SK10153 (thyA715). SK9124 [rph-1 thyA715/pBMK11(pcnB+/CmR)] and SK10148 (Drnt::kan rph-1 thyA715) have been previously described (2,24). A P1 lysate grown on SK10148 was used to transduce SK10153 (thyA715) and SK10019 (pnpD683 rph-1 thyA715) to construct SK10592 (Drnt::kan thyA715) and SK10609 (Drnt pnpD683 rph-1 thyA715), respectively. SK10575 (Drnb Drnt rph-1 thyA715) was made by transduction with P1 phage grown on CMA201 (Drnb::tetR rph-1 thyA715) (29) into SK10148 (Drnt::kan rph-1 thyA715). The DpcnB::aac(3)-IV (apramycin, AprR) deletion/substitution allele in SK4465 was obtained using the method of Hamilton et al. (30). Briefly, the pcnB coding sequence starting from amino acid six after the UUG translation start codon until two amino acids upstream of the translation stop codon was replaced by the aac(3)-IV apramycin resistance cassette obtained from plasmid pSET152 (Genbank Accession No. 414670). SK10593 [Drnt DpcnB::aac(3)-IV] and SK10020 (...truncated)