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Elevated Levels of Era Gtpase Improve Growth, 16S Rrna Processing, and 70S Ribosome Assembly of Escherichia Coli Lacking Highly

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Elevated Levels of Era Gtpase Improve Growth, 16S Rrna Processing, and 70S Ribosome Assembly of Escherichia Coli Lacking Highly Elevated levels of Era GTPase improve growth, 16S rRNA processing, and 70S ribosome assembly of Escherichia coli lacking highly conserved multifunctional YbeY endoribonuclease The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Ghosal, Anubrata, Vignesh M.P. Babu, and Graham C. Walker. "Elevated Levels of Era GTPase Improve Growth, 16S rRNA Processing, and 70S Ribosome Assembly of Escherichia coli Lacking Highly Conserved Multifunctional YbeY Endoribonuclease." Journal of Bacteriology, 200, 17 (August 2018) e00278-18. As Published http://dx.doi.org/10.1128/jb.00278-18 Publisher American Society for Microbiology Version Author's final manuscript Citable link https://hdl.handle.net/1721.1/123979 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/ 1 Elevated levels of Era GTPase improve growth, 16S rRNA processing, and 2 70S ribosome assembly of Escherichia coli lacking highly conserved 3 multifunctional YbeY endoribonuclease 4 5 Anubrata Ghosal, Vignesh M.P. Babu and Graham C. Walker* 6 7 Department of Biology, Massachusetts Institute of Technology, 8 Cambridge, MA 02139 9 USA 10 11 12 13 * Corresponding author. E-mail address: [email protected] (G.C. Walker). 14 15 Keywords: YbeY, Era, 16S rRNA, ribosome, and E. coli. 16 17 18 19 20 21 22 23 1 24 Abstract 25 YbeY is a highly conserved, multifunctional endoribonuclease that plays a significant role in 26 ribosome biogenesis and has several additional roles. Here, we show in Escherichia coli that 27 overexpressing the conserved GTPase, Era, partially suppresses the growth defect of a ΔybeY strain 28 while improving 16S rRNA processing and 70S ribosome assembly. This suppression requires 29 both Era’s ability to hydrolyze GTP and the function of three exoribonucleases, RNase II, RNase 30 R and RNase PH, suggesting a model for Era’s action. Overexpressing Vibrio cholerae Era 31 similarly partially suppresses the defects of an E. coli ΔybeY strain indicating this property of Era 32 is conserved in bacteria other than E. coli. 33 Importance 34 This work provides additional insights into the critical, but still incompletely understood, 35 mechanism of the processing of the E. coli 16S rRNA 3’-terminus. The highly conserved GTPase, 36 Era, is known to bind to the precursor of the 16S rRNA near its 3-end. Both the endoribonuclease 37 YbeY, which binds to Era, and four exoribonucleases have been implicated in this 3’-end 38 processing. Results reported here offer additional insights into the role of Era in 16S rRNA 3’- 39 maturation and into the relationship between the action of the endoribonuclease YbeY and the four 40 exoribonucleases. This study also hints at why YbeY is only essential in some bacteria and 41 suggests that the YbeY could be a target for a new class of antibiotic in these bacteria. 42 43 44 45 2 46 Introduction 47 Ribosome biogenesis is a key complex cellular event involving multiple steps of synthesis, 48 processing, and modification and is conserved in all living organisms. The various processes in 49 ribosome biogenesis do not always occur sequentially but rather some start before others. 50 Individual steps are regulated at multiple levels, and defects in any of these steps, such as 16S 51 rRNA processing, can impact ribosome structure and the overall level of proteins in the cell (1, 2). 52 The initiation of ribosome biogenesis starts with the transcription of rRNA genes. The 53 Escherichia coli genome has seven rRNA operons, rrnD, rrnG, rrnH, rrnC, rrnA, rrnB and rrnE 54 (3). Genes in each rrn operons are transcribed together as a polycistronic RNA, which is 55 subsequently processed into the mature 16S, 23S, and 5S rRNAs, which are 1542, 2904, and 120 56 nucleotides long, respectively (4–6). The processing of rRNA starts before the completion of 57 transcription of rRNA genes. The endoribonuclease, RNase III, acts first on rRNA transcripts and 58 separates rRNA precursors, which are then processed further by ribonucleases to their mature 59 forms (4). RNase E acts on the 5’-end of immature 16S rRNA, leaving 66 nucleotides that are then 60 removed by RNase G processing. Together, RNase E and G remove 115 nucleotides from the 5’- 61 end of immature 16S rRNA (7). The complexity of the 16S processing is highlighted in a recent 62 study which has revealed that the dominant pathways entail complete processing of 5’-end prior 63 to the 3’-end maturation (8). Sulthana et al. show that the four 3’-to-5’-exoribonucleases, RNase 64 II, RNase PH, RNase R and PNPase, contribute significantly in the processing of the 3’-terminus 65 of 16S rRNA that removes the extra 33 nucleotides from the 3’-end (9). 66 A striking defect in the processing of 16S rRNA has been observed in E. coli lacking an 67 extremely highly conserved, multifunctional gene ybeY (10), which encodes a single-strand- 68 specific endoribonuclease (8, 11). Although there is a minor defect in the processing of the 5’-end 3 69 of 16S RNA in a ΔybeY strain, the 3’-processing of 16S rRNA is the most strongly affected (10). 70 Thus, the ΔybeY strain produces only a limited amount of fully processed 16S rRNA and 71 accumulates unprocessed 17S rRNA and truncated 16S rRNA, 16S*(11). Northern analysis 72 confirms that unprocessed 17S rRNA have defined immature 5’- and 3’-termini (10), and almost 73 all the 16S* rRNAs lack the 3’-terminus of 16S rRNA whereas the 5’-terminus is present nearly in 74 all the 16S* rRNAs (11). Both the proper YbeY-dependent processing of the 3’-terminus of 16S 75 rRNA in vivo and YbeY’s single-strand-specific endoribonuclease activity in vitro require the 76 conserved His114 and R59 residues located in the presumed catalytic site (10, 11). Furthermore, 77 in the absence of YbeY, cells produce many defective 30S ribosomal subunits and show defects 78 in the assembly of 70S ribosomes (10, 11). Additional studies have revealed that YbeY binds to 79 16S rRNA (12) and YbeY, together with an exonuclease RNase R, is also involved in 70S 80 ribosomal quality control. A model has been suggested for how YbeY might participate together 81 with exoribonucleases in the processing of 3’-end of 16S rRNA (11). 82 Recently, we have presented evidence that YbeY interacts directly with ribosomal protein 83 S11 and with the GTPase Era, which binds near the 3’-end of the 16S rRNA precursor (13). Our 84 data suggest that these interactions position YbeY on the ribosome, thereby potentially allowing 85 YbeY to cleave the 16S rRNA precursor, 17S rRNA (13). The GTPase domain of Era protein 86 interacts with YbeY, whereas the opposite side of the catalytic domain of YbeY interacts with 87 S11(13). GTPases, RNA helicases and RNA chaperones are involved in a variety of steps of 88 ribosome biogenesis. These include recovering rRNA from unfavorable intermediate forms, 89 displaying rRNA for accurate processing, triggering cellular signals, and facilitating assembly of 90 ribosomal proteins (2). 4 91 Era is known to be a key ribosome-associated GTPase that plays major roles in 16S rRNA 92 processing (14). Era interacts with 16S rRNA and pre-30S ribosomal subunit (15, 16). The GTP- 93 bound form of Era binds near the 3’-end of unprocessed 16S rRNA between nucleotides 1531 and 94 1539, thereby leaving the ultimate 3’-terminus (1542) and the extra 33 nucleotides exposed (16). 95 Once 16S RNA processing is completed, Era hydrolyzes GTP and leaves the mature 16S rRNA 96 and thus has been proposed to act both as a chaperone for processing and maturation of 16S rRNA 97 and as a checkpoint for assembly of the 30S ribosomal subunit (16). At lower than the normal 98 physiological level of Era, cells accumulate 16S rRNA precursors and also unassembled 30S and 99 50S ribosomal subunits (17). 16S rRNA precursors also accumulate in the absence of RbfA (18), 100 which is a ribosome maturation factor that binds to the 30S ribosomal subunit (19). Overexpression 101 of Era can compensate for the loss of RbfA (17). This indicates that overexpression of one 102 ribosomal factor could potentially compensate for the loss of another ribosomal factor. In support 103 of this idea, elevating the level of RbfA suppresses the 16S rRNA processing defects observed in 104 a strain lacking a ribosome maturation factor, RimM (18), and, structurally, RbfA is similar to the 105 KH domain of Era (20). Therefore, in this study, we have tested if overexpression of Era can 106 compensate for the loss of YbeY. 107 Results and Discussion 108 Overexpression of Era improves growth of the ∆ybeY strain and also 16S rRNA 109 processing. The observations described above (17–19) stimulated us to investigate whether 110 elevating the level of Era might suppress the loss of YbeY function by improving the processing 111 defects of 16S rRNA. We, therefore, cloned the era gene in a plasmid under the control of a 112 tetracycline promoter and transformed the resulting Era plasmid, pEra, into a ΔybeY strain. The 113 overexpression strain, ΔybeY pEra, produced ~15 times more Era transcript compared to the 5 114 wildtype strain containing empty pASK-IBA3C plasmid, wildtype pCon, (Fig. 1a). Analysis of the 115 growth of a ∆ybeY pEra strain showed that the elevated level of Era improved the growth of a 116 ΔybeY strain significantly, but that the ΔybeY pEra strain still grew slower than the wildtype pCon 117 strain (Fig. 1b). This result indicates that elevating the level of Era does not completely rescue all 118 the defects of a ΔybeY strain.
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