Bacillus subtilis trp Leader RNA: RNase J1 endonuclease cleavage specificity and PNPase processing

Abstract

In the presence of ample tryptophan, transcription from the Bacillus subtilis trp operon promoter terminates to give a 140-nucleotide trp leader RNA. Turnover of trp leader RNA has been shown to depend on RNase J1 cleavage at a single-stranded, AU-rich region just upstream of the 3' transcription terminator. The small size of trp leader RNA and its strong dependence on RNase J1 cleavage for decay make it a suitable substrate for analyzing the requirements for RNase J1 target site specificity. trp leader RNAs with nucleotide changes around the RNase J1 target site were more stable than wild-type trp leader RNA, showing that sequences on either side of the cleavage site contribute to RNase J1 recognition. An analysis of decay intermediates from these mutants suggested limited 3'-to-5' exonuclease processing from the native 3' end. trp leader RNAs were designed that contained wild-type or mutant RNase J1 targets elsewhere on the molecule. The presence of an additional RNase J1 cleavage site resulted in faster RNA decay, depending on its location. Addition of a 5' tail containing 7 A residues caused destabilization of trp leader RNAs. Surprisingly, addition at the 5' end of a strong stem loop structure that is known to stabilize other RNAs did not result in a longer trp leader RNA half-life, suggesting that the RNase J1 cleavage site may be accessed directly. In the course of these experiments, we found evidence that polynucleotide phosphorylase processivity was inhibited by a GCGGCCGC sequence.

FIGURE 1.

A, schematic diagram of trp leader RNA, showing structural features and location of primary RNase J1 cleavage site. B, Northern blot analysis of trp leader RNA in wild-type and RNase J1 conditional mutant strains grown in the presence of 1 mm IPTG. Time after rifampicin addition (min) is indicated above each lane. Migration of the 136-nt marker (lane M) is indicated at left. Semi-log plot of % RNA remaining versus time is shown at right. Open triangles, wild type; closed squares, RNase J1 mutant.

FIGURE 2.

A, trp leader RNA sequence, showing mutations in the RNase J1 target site. Extent of complementarity of 5′ end-labeled probes to sequences near the 5′ and 3′ ends is indicated. Below the trp leader RNA schematic is shown the wild-type and mutant sequences of the RNase J1 target site. Half-lives of each RNA (min) are shown. The major site of RNase J1 cleavage after nt 101 is indicated by the arrowhead. B–D, Northern blot analysis of wt and mutant trp leader RNA, using probe 1 on low-resolution (B) and high-resolution (D) blots, and probe 2 (C). FL, full-length trp leader RNA. Leftmost lane (M) in B and C contained 5′ end-labeled TaqI fragments of plasmid pSE420 DNA (35), and the sizes of these fragments are indicated on the left. Sequencing ladder in D was generated on M13mp18 single-stranded DNA. Sizes of RNA bands detected by probe 1 in D and discussed in the text are indicated on the right. In B and D, the 101-nt upstream product of RNase J1 cleavage is indicated by the caret.

FIGURE 3.

trp leader RNA mutant constructs. Open rectangle denotes RNase J1 target site, nt 96–103. Filled rectangle denotes target site mutated to contain the NotI sequence. The asterisk in RNAs A5, B5, and C5 indicates accumulation of small decay intermediate consisting of 5′ proximal sequence. Half-life of each RNA (average of at least three independent experiments, with S.D.) is shown to the right of the schematic. ND, not done.

FIGURE 4.

A, half-life of ∼50-nt processing intermediate (arrow) in the RNA with the upstream NotI site (RNA A5). B, sizing of the ∼50-nt RNA on a high-resolution gel by Northern blot analysis of in vivo RNA isolated from strain carrying RNA A5 (left) and after PNPase processing in vitro (right). The in vitro samples were incubated without (−) or with (+) PNPase. C, comparison of amount of full-length (FL) and ∼50-nt processing intermediate (arrow) in A5 and A6 RNAs. D, in vitro PNPase activity on trp leader RNAs without and with the upstream NotI sequence. Sizes of full-length substrates (114 nt for wild-type, 127 nt for RNA A5) are indicated, as well as the approximate size of the PNPase digestion product for the RNA with the upstream NotI sequence. The control lane C contained no added PNPase. Above each lane is indicated time (min) after PNPase addition.

FIGURE 5.

Steady state pattern of selected trp leader RNAs. Full-length (FL) and processed products detected by probe 1 in strains carrying the construct indicated above each lane.