Distributive enzyme binding controlled by local RNA context results in 3′ to 5′ directional processing of dicistronic tRNA precursors by<i>Escherichia coli</i>ribonuclease P

Zhao, Jing; Harris, Michael E.

doi:10.1093/nar/gky1162

Cited by 6 publications

(9 citation statements)

References 107 publications

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“…In contrast to RNase E where endonucleolytic cleavages in A/U rich regions of the single‐stranded RNAs (McDowall et al., 1994, 1995) can proceed either in the 5' → 3' direction (Richards & Belasco, 2019) or by a direct entry mechanism (Clarke et al., 2014; Garrey & Mackie, 2011; Kime et al., 2014), RNase P endonucleolytic cleavages proceed in the 3' → 5' direction, both in vivo and in vitro, based on the analysis of tRNA substrates (Agrawal et al., 2014; Zhao & Harris, 2019). Furthermore, the RNase P holoenzyme from both E. coli and Bacillus subtilis has been shown to cleave single‐stranded RNA in vitro with some sequence preferences, although much less efficiently compared with the structured RNAs (Hansen et al., 2001; Loria & Pan, 2000).…”

Section: Discussionmentioning

confidence: 99%

Inactivation of RNase P in Escherichia coli significantly changes post‐transcriptional RNA metabolism

Mohanty

Kushner

2021

Molecular Microbiology

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Ribonuclease P (RNase P), which is required for the 5'‐end maturation of tRNAs in every organism, has been shown to play a limited role in other aspects of RNA metabolism in Escherichia coli. Using RNA‐sequencing (RNA‐seq), we demonstrate that RNase P inactivation affects the abundances of ~46% of the expressed transcripts in E. coli and provide evidence that its essential function is its ability to generate pre‐tRNAs from polycistronic tRNA transcripts. The RNA‐seq results agreed with the published data and northern blot analyses of 75/83 transcripts (mRNAs, sRNAs, and tRNAs). Changes in transcript abundances in the RNase P mutant also correlated with changes in their half‐lives. Inactivating the stringent response did not alter the rnpA49 phenotype. Most notably, increases in the transcript abundances were observed for all genes in the cysteine regulons, multiple toxin‐antitoxin modules, and sigma S‐controlled genes. Surprisingly, poly(A) polymerase (PAP I) modulated the abundances of ~10% of the transcripts affected by RNase P. A comparison of the transcriptomes of RNase P, RNase E, and RNase III mutants suggests that they affect distinct substrates. Together, our work strongly indicates that RNase P is a major player in all aspects of post‐transcriptional RNA metabolism in E. coli.

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Section: Discussionmentioning

confidence: 99%

Inactivation of RNase P in Escherichia coli significantly changes post‐transcriptional RNA metabolism

Mohanty

Kushner

2021

Molecular Microbiology

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“…but not the alaX pre-tRNAs with unprocessed 5′-leader sequences (the alaW and alaX intergenic sequences), in the strain containing the wild type RNase P (Figure 2b, lane 2; Figure 5, lane 2-3) indicated that the initial RNase P cleavage occurred at the alaX 5′-mature end separating the two pre-tRNAs following the removal of the Rho-independent transcription terminator by RNase E. The second RNase P cleavage occurred at the 5′-mature ends of alaW pre-tRNAs following the removal of its 3′ trailer sequences suggesting that the RNase P cleavage of alaW alaX transcript proceeds in a 3′ → 5′ direction (Figure 11). The 3′ → 5′ directionality of RNase P cleavages has been documented previously both in vivo (Agrawal et al, 2014) and in vitro (Zhao & Harris, 2019).…”

Section: Discussionmentioning

confidence: 53%

Processing of the alaW alaX operon encoding the Ala2 tRNAs in Escherichia coli requires both RNase E and RNase P

Mohanty

Kushner

2022

Molecular Microbiology

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The 86 tRNAs in Escherichia coli are transcribed either as mono-or polycistronic transcripts, which are often part of operons containing either mRNAs or rRNAs (Mohanty & Kushner, 2019b). Since all tRNAs are transcribed as precursors (pre-tRNAs), they require extensive processing at both their 5′ and 3′ ends to become functional. At least seven distinct tRNA processing pathways have been identified that account for the maturation of 31/86 of the tRNAs in E. coli (Mohanty & Kushner, 2019b. All tRNA processing pathways require RNase P, a ribonucleoprotein, which consists of a small protein component (C5 protein encoded by rnpA) and a single RNA subunit (M1 RNA encoded by rnpB) that is responsible for the maturation of the 5′-ends of each tRNA. The enzyme is also required for the separation of pre-tRNAs from nine polycistronic tRNA transcripts (Mohanty & Kushner, 2022). E. coli contains two distinct isotypes of alanine tRNAs, Ala1B andAla2. The Ala1B tRNAs, encoded by alaT, alaU and alaV recognize the GCU, GCA and GCG codons, while the Ala2 tRNAs, encoded by alaW and alaX, recognize the GCC codon. Recently, the maturation of the Ala1B tRNA isotypes including the Glu2, Ile1 tRNAs, which are embedded within the seven rRNA operons has been described . However, the maturation pathway for the Ala2 tRNAs at the molecular level has not been determined. The genomic organization of alaW and alaX genes suggests that they form a dicistronic operon that is not associated with the rRNA operons (Keseler et al., 2021) (Figure 1). The tRNA sequences are separated by a 39 nt spacer and include a Rho-independent transcription terminator 7 nt downstream of alaX CCA determinant (Figure 1).Our previous transcriptome analyses using RNA-seq showed that the level of the alaW alaX transcripts increased in a rnpA49 mutant (Mohanty & Kushner, 2022) suggesting a role of RNase P in their separation as well as maturation. Surprisingly, unprocessed alaW alaX RNA levels increased dramatically in a rnpA49 ΔpcnB double mutant compared to the rnpA49 single mutant, indicating a role

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“…Nonetheless, there is evidence that structural anti-determinants play a role in polycistronic ptRNA processing by E. coli RNase P. Remarkably, all seven valine tRNAs in E. coli require RNase P for separation from their primary polycistronic transcripts, which processes them in a 5′ to 3′ directional manner 59 . In the valVW operon a stem-loop in 5′ leader of tRNA valV inhibits RNase P cleavage resulting in enhanced cleavage of the 3’ most tRNA valW thus enforcing directional processing 24 , 60 . In the valU operon, the most 5′ ptRNA valU is processed last and has GG at N(−1)N(−2), the internal tRNA valX and tRNA valY have GU, while the 3′ tRNA lysV that is processed first has a near optimal UC dinucleotide.…”

Section: Discussionmentioning

confidence: 99%

“…1a) 3,8 . These structural anti-determinants can involve the formation of stem loops in the 5′ leader 19,24 or pairing between the 3′ RCCA of tRNA and N(−1) to N(−3) 27,35,36 . To comprehensively identify and parse sequence determinants and anti-determinants in the 5′ leader, we used highthroughput sequencing kinetics (HTS-Kin) to measure the relative k cat /K m for all possible combinations of 5′ leader nucleotides N(−6) to N(−1) (n = 4096) [19][20][21]37 .…”

Section: Comprehensive Identification Of Ptrna 5′ Leader Determinants...mentioning

confidence: 99%

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Structural and mechanistic basis for recognition of alternative tRNA precursor substrates by bacterial ribonuclease P

et al. 2022

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Binding of precursor tRNAs (ptRNAs) by bacterial ribonuclease P (RNase P) involves an encounter complex (ES) that isomerizes to a catalytic conformation (ES*). However, the structures of intermediates and the conformational changes that occur during binding are poorly understood. Here, we show that pairing between the 5′ leader and 3′RCCA extending the acceptor stem of ptRNA inhibits ES* formation. Cryo-electron microscopy single particle analysis reveals a dynamic enzyme that becomes ordered upon formation of ES* in which extended acceptor stem pairing is unwound. Comparisons of structures with alternative ptRNAs reveals that once unwinding is completed RNase P primarily uses stacking interactions and shape complementarity to accommodate alternative sequences at its cleavage site. Our study reveals active site interactions and conformational changes that drive molecular recognition by RNase P and lays the foundation for understanding how binding interactions are linked to helix unwinding and catalysis.

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Distributive enzyme binding controlled by local RNA context results in 3′ to 5′ directional processing of dicistronic tRNA precursors byEscherichia coliribonuclease P

Cited by 6 publications

References 107 publications

Inactivation of RNase P in Escherichia coli significantly changes post‐transcriptional RNA metabolism

Inactivation of RNase P in Escherichia coli significantly changes post‐transcriptional RNA metabolism

Processing of the alaW alaX operon encoding the Ala2 tRNAs in Escherichia coli requires both RNase E and RNase P

Structural and mechanistic basis for recognition of alternative tRNA precursor substrates by bacterial ribonuclease P

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