The Physiological Role of RNase T Can Be Explained by Its Unusual Substrate Specificity

Abstract

Escherichia coli RNase T, the enzyme responsible for the end-turnover of tRNA and for the 3 maturation of 5 S and 23 S rRNAs and many other small, stable RNAs, was examined in detail with respect to its substrate specificity. The enzyme was found to be a single-strandspecific exoribonuclease that acts in the 3 to 5 direction in a non-processive manner. However, although other Escherichia coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair. The presence of a free 3-hydroxyl group is required for the enzyme to initiate digestion. Studies with RNA homopolymers and a variety of oligoribonucleotides revealed that RNase T displays an unusual base specificity, discriminating against pyrimidine and, particularly, C residues. Although RNase T appears to bind up to 10 nucleotides in its active site, its specificity is defined largely by the last 4 residues. A single 3-terminal C residue can reduce RNase T action by >100-fold, and 2-terminal C residues essentially stop the enzyme. In vivo, the substrates of RNase T are similar in that they all contain a doublestranded stem followed by a single-stranded 3 overhang; yet, the action of RNase T on these substrates differs. The substrate specificity described here helps to explain why the different substrates yield different products, and why certain RNA molecules are not substrates at all.RNase T, one of eight exoribonucleases present in Escherichia coli (1), was originally identified as an activity involved in the end-turnover of tRNA (2, 3). This process consists of the removal and re-addition of the 3Ј-terminal AMP and, to a very small extent, the penultimate CMP residues of tRNA. Other studies demonstrated that RNase T also is involved in other aspects of RNA metabolism, including the 3Ј maturation of tRNAs (4), 5 S and 23 S rRNAs (5, 6), and other small, stable RNAs (7). Although multiple exoribonucleases may contribute to the 3Ј maturation of these RNAs, RNase T is often the most efficient (8). In fact, RNase T is essential for generating the mature 3Ј-ends of 5 S and 23 S rRNAs (5, 6). Interestingly, all the RNase T substrates share a common sequence feature, i.e. their 5Ј-and 3Ј-ends pair with each other to form a stable, double-stranded (ds) 1 stem followed by a few unpaired 3Ј-nucleotides (7). 5 S and 23 S rRNAs differ from the other small, stable RNAs in that their mature forms contain only 1 or 2 unpaired 3Ј residues, whereas the others contain 4 unpaired residues when matured. Thus, RNase T appears to be the only exoribonuclease that can efficiently remove residues near a stable double-stranded stem. However, what determines the number of unpaired residues remaining after RNase T action is still unknown.In vitro, purified RNase T acts on a variety of tRNA-like substrates (2, 9). Of these, the preferred substrate is mature tRNA-CCA, but terminal residues also can be removed from phosphodiesterase-treated tRNA and removed slowly from tRNA-CA and tRNA-CCA-C 2-3 . In contrast, tRNA-CCp, aminoacyl...

Mechanism of Action of RNase T

“…We reasoned that if the NBS residues were distant enough from the catalytic center, they might be required for action on tRNA, but not for the binding of very short substrates. The DNA oligonucleotides dA4 and dA2 were used for these studies rather than RNA oligonucleotides because DNA substrates have considerably lower K m values (10,11). As shown in Table IV, for NBS1 mutant R15A and the NBS2/NBS3 triple mutant K108A/R112A/K139A, alanine substitutions have virtually no effect on RNase T activity against the short oligonucleotides, in contrast to the major loss in activity when tRNA was the substrate (Table III).…”

“…Although the substrates of RNase T in vivo share a common sequence feature consisting of a stable, double-stranded (ds) stem followed by a few unpaired 3Ј nucleotides, RNase T actually is a single-strand-specific exoribonuclease that acts in the 3Ј-to-5Ј direction in a non-processive manner (11). However, whereas other E. coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair, although it slows as it approaches the duplex structure.…”

“…However, whereas other E. coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair, although it slows as it approaches the duplex structure. The presence of a free 3Ј-hydroxyl group is required for the enzyme to initiate digestion (11).…”

Structure and function of the polymerase core of TRAMP, a RNA surveillance complex

“…RNA ends have also been found to be recognition elements for other proteins involved in noncoding RNA processing and/or surveillance, including T455 I175 S176 V374 I377 D177 N371 I366 E378 D367 D365 R360 T363 P359 H358 E381 N386 K444 R441 M357 Y403 V402 F404 G388 V392 L382 F354 D391 D390 L394 I308 V146 I144 Y143 A142 R141 W140 R135 I139 L198 N197 F193 S191 N197 the eukaryotic proteins La and Ro (19,20), which both bind nascent transcripts, as well as the bacterial RNase T and RNase R (21,22). Sequences at the 3′ end may be one mechanism for recognizing such transcripts.…”

Repairing tRNA termini: News from the 3' end

“…36 This points to another function of the enzyme, apart from the de novo synthesis of the CCA end: i.e., the repair of partially degraded CCA ends. In E. coli, 3 0 end turnover of RNA is mainly catalyzed by RNase T. tRNA-degradation intermediates would be expected to be mostly lacking the terminal adenosine, since RNase T activity is biased against C. [37][38][39] Indeed, the E. coli CCA-adding enzyme was shown to accept even a minimal CpC substrate for adenosine addition. This ability of the enzyme to restore the CCA from intermediates allows the efficient repair of tRNAs lacking the terminal A.…”