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 conserved multifunctional YbeY endoribonuclease

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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

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

5 Anubrata Ghosal, Vignesh M.P. Babu and Graham C. Walker*

7 Department of Biology, Massachusetts Institute of Technology,

8 Cambridge, MA 02139

9 USA

13 * Corresponding author. E-mail address: [email protected] (G.C. Walker).

15 Keywords: YbeY, Era, 16S rRNA, ribosome, and E. coli.

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.

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

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).

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

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.

119 Since the 16S RNA processing defect likely contributes to the slow growth of the ΔybeY

120 strain, we examined whether the elevated level of Era was reducing its 16S rRNA processing

121 defect. We observed that the amount of mature 16S rRNA was increased in ΔybeY pEra strain as

122 compared to the ΔybeY strain carrying the empty plasmid, ΔybeY pCon (Fig. 1c). Furthermore, the

123 amount of 16S* rRNA was reduced significantly in the ΔybeY pEra strain compared to the ΔybeY

124 pCon strain and there was a slight reduction in the amount of immature 17S rRNA as well. These

125 results are consistent with the previously reported critical role of Era in the processing of 17S

126 rRNA. Since RbfA is known to participate in the 5’-end maturation of 16S rRNA (18) and a ΔybeY

127 mutant has a minor defect in 5’-end processing (10), we also tried overexpressing RbfA and

128 observed that elevated RbfA also slightly improved the growth defect and 16S rRNA processing

129 defect of ΔybeY strain (Fig. S1).

130 Overexpression of Era improves the 70S ribosome assembly in the ∆ybeY strain.

131 Several studies have shown that strains having defects in 16S rRNA processing also exhibit

132 defective 70S ribosome assembly (10, 17). We hypothesized that improved 16S processing caused

133 by the elevated level of Era might also partially reduce the 70S ribosome assembly defect of the

134 ΔybeY strain. Consistent with this expectation, we observed that the ΔybeY pEra strain exhibited a

135 reduction in the amounts of unassembled 30S and 50S ribosomal subunits and showed a

136 concomitant increase of 70S ribosomes as compared to the ΔybeY pCon strain (Fig. 1d). Although

137 the amount of 70S ribosome obtained from the 70S fraction of the ΔybeY pEra strain in this

138 experiment is similar to wildtype, the ΔybeY pEra strain had markedly less 70S ribosomes in the

139 translating polysome fraction compared to wildtype. The failure to completely recover 70S

140 ribosome assembly upon Era overexpression might explain the slower growth of ΔybeY pEra strain

141 in comparison to the wildtype pCon strain.

142 Overexpression of Era does not rescue the sensitivity of ∆ybeY strain to various stresses.

143 Previous studies on E. coli and other bacteria have shown that, in addition to its role in rRNA

144 processing required for ribosome biogenesis, YbeY affects 70S ribosome quality control, both

145 Hfq–dependent and –independent small RNA regulation, stress regulation, symbiosis, and

146 virulence and the loss of YbeY results in sensitivity to different stresses (11, 13, 21–24). We

147 therefore investigated whether an elevated level of Era can improve the growth of ΔybeY strain

148 under various stressful conditions. We analyzed the growth of ΔybeY pEra strain along with ΔybeY

149 pCon and wildtype pCon strains in response to antibiotics that either target the 30S ribosomal

150 subunit (tetracycline and kasugamycin) or cell wall (ampicillin), as well as to heat and two types

151 of DNA damaging agents (UV radiation and nitrofurazone). In contrast to the effect of an elevated

152 level of Era on 16S rRNA processing and ribosome assembly, we did not observe any significant

153 improvement of growth of ΔybeY pEra strain compared to the ΔybeY pCon strain response to these

154 different stresses (Fig. 1e). This observation suggests that the amount of translating polysomes in

155 ΔybeY pEra strain is insufficient for survival in the presence of external stress. The elevated level

156 of Era slightly sensitized the ΔybeY strain to kasugamycin and tetracycline (Fig. 1e), an effect that

157 might possibly be due to a direct interaction between Era and the RsmA (KsgA) methyltransferase

158 or to an effect on the conformation of 16S rRNA (25). Both kasugamycin and tetracycline inhibit

159 translation by blocking the binding of tRNA to mRNA-30S ribosomal complex (26, 27), so they

160 may respond similarly to the change caused by elevated levels of Era. Alternatively, it is possible

161 that the ΔybeY pEra strain might have improved 70S assembly even though it still has considerable

162 16S rRNA processing defect. This might result in the defective ribosome assembly with immature

163 rRNA leading to sensitivity to 30S targeting antibiotics.

164 GTPase activity of Era is required for suppressing the ΔybeY growth defect. A

165 previous study has demonstrated that the Era mutant (P17R), which has reduced GTPase activity,

166 is capable of suppressing the function of dnaG in DNA replication, leading to the speculation that

167 the reduction of the Era GTPase slows down or blocks the cell cycle progression, thereby allowing

168 extra time to repair damaged DNA (28). We used this same mutant to evaluate the role of Era’s

169 GTPase activity in suppressing the growth defect of ΔybeY. The EraP17R overexpression strain,

170 ΔybeY(peraP17R), grew more slowly than the ΔybeY pEra strain (Fig. 2), indicating that Era needs

171 to be able to hydrolyze GTP in order to suppress the functional loss of YbeY. It is possible that the

172 less catalytically active Era remains bound to the mature 16S rRNA much longer than the wild

173 type Era, thereby delaying the formation of 70S ribosomes (16), or alternatively that it might bind

174 to rRNA more slowly (28).

175 Overexpression of Era increases 16S rRNA by improving processing rather than

176 stability. We then investigated whether the increased amount of mature 16S rRNA in the ΔybeY

177 pEra strain is due to the synthesis of more 16S rRNA or whether an elevated level of Era provides

178 stability to 16S rRNA. Total RNA was analyzed from rifampicin treated and non-treated samples.

179 The amount of 16S* recovered from both ΔybeY strains in this experiment were higher compared

180 to figure 1 due to different sample processing method (see Materials and methods). Firstly, in the

181 absence of rifampicin, the amount of 16S rRNA was higher in ΔybeY pEra strain compared to the

182 ΔybeY pCon strain (Fig. 3, left panel). On the other hand, no substantial difference was observed

183 in the 16S rRNA amount between ΔybeY pCon and ΔybeY pEra strains in the presence of rifampicin

184 (Fig. 3, Right panel). When transcription was occurring, an elevated level of Era improved the

185 conversion of 17S rRNA precursors to 16S rRNA, suggesting that overexpression of Era increases

186 16S rRNA by improving processing. Interestingly, we observed that when transcription was

187 blocked due to the addition of rifampicin, neither the ΔybeY pEra nor the ΔybeY pCon strains, were

188 able to retain 16S rRNA. Instead, the majority of 16S rRNA was converted to 16S* rRNA,

189 suggesting the inability of Era and additional direct or indirect roles of YbeY in providing stability

190 to the mature 16S.

191 Suppression of growth and 16S processing in ΔybeY strain requires three

192 exoribonucleases and is partially dependent on PNPase. We hypothesized that the previously

193 reported four exoribonucleases that participate in the processing of 3’-end of 16S rRNA in wild

194 type cells would be important for the Era-mediated suppression of the 16S rRNA processing

195 defect. Each of these four exoribonucleases has been shown to partially contribute to the 3’-end

196 processing of 16S rRNA in a ybeY+ strain background in a redundant fashion (9). Consistent with

197 this, a 3’-end processing defect of 16S rRNA was not observed in the absence of RNase R (Δrnr),

198 RNase PH (Δrph), or PNPase (Δpnp) (10). Either RNase II or RNase R have been observed to be

199 more efficient in completing the 3’-end processing of 16S rRNA when the other three exonucleases

200 are absent than are PNPase or RNase PH (9). Interestingly, RNase R and RNase II can both utilize

201 substrates having a 3’-phosphate group (29), the type of terminus produced by YbeY cleavage

202 (11). Strikingly, we found that excess Era was not able to improve the growth and 16S rRNA

203 processing defects in the double knockout strains ΔybeY Δrnb, ΔybeY Δrnr, and ΔybeY Δrph (Fig.

204 4a-c), which lack RNase II, RNase R and RNase PH respectively in addition to YbeY, and was

205 only partially able to do so in ΔybeY Δpnp strain, which lacks PNPase (Fig. 4d). Thus, the Era-

206 mediated suppression of growth and 16S rRNA processing reported in this study depends almost

207 completely on the combined action of RNase II, RNase R and RNase PH, while the dependency

208 on PNPase seems more limited. This result suggests that all four exoribonucleases participate in

209 the 3’-end processing of 16S rRNA in the absence of YbeY, which contrasts the functional

210 redundancy of these exoribonucleases in the ybeY+ strain. A similar requirement for multiple

211 exoribonucleases for 3’-end maturation of 16S rRNA in a ΔybeY mutant is seen even without Era

212 overproduction since even the modest level of correctly matured 16S rRNA observed in a ΔybeY

213 mutant is greatly reduced by deleting either rnr or pnp [9].

214 Overexpression of V. cholerae Era improves growth of an E. coli ∆ybeY strain and

215 also 16S rRNA processing. YbeY is present in the pathogen Vibrio cholerae, where it is essential

216 and plays roles in ribosome biogenesis, small RNA regulation, stress regulation and pathogenicity

217 (23). As in E. coli, the loss of YbeY function in V. cholerae affects 16S RNA maturation including

218 the processing of 3’-end, 70S ribosome assembly, and small RNA regulation (23). Moreover, the

219 biochemical properties of the purified V. cholerae YbeY endoribonuclease are identical to those

220 of E. coli YbeY (23). Since Era is also a highly conserved protein (14), we tested whether elevated

221 levels of V. cholerae Era could suppress the growth and 16S rRNA processing deficiencies of an

222 E. coli ΔybeY strain. We observed that, despite V. cholerae Era only having ~65% amino acid

223 sequence similarity to E. coli Era (Fig. S2a), it was able to significantly improve the growth and

224 16S rRNA processing in ΔybeY strain (Fig. S2b and S2c), suggesting that Era’s role in 16S rRNA

225 processing is conserved in Vibrio cholerae.

226 Conclusions

227 Our results suggest a simple model (Fig. 5a) in which elevating the level of Era increases

228 the binding of GTP-bound Era to the unprocessed 3’-terminus of the 16S rRNA precursor present

229 in the pre-30S ribosomal complexes. The three exoribonucleases RNase R, RNase PH and RNase

230 II, with a contribution from PNPase, all act together to remove the 33 precursor nucleotides from

231 the 3’-terminus of 16S rRNA while it is bound to Era-GTP, with the extent of the processing being

232 sterically limited by the Era protein. Following exoribonuclease processing, GTP hydrolysis

233 releases Era from the mature 16S rRNA. Since Era and YbeY interact and YbeY also interacts

234 with the S11 protein (13), perhaps the elevated levels of Era partially compensate for the loss of

235 important protein-protein interactions normally provided by YbeY. Another possibility is that the

236 interaction of YbeY with Era increases the rate of GTP hydrolysis by Era after 16S rRNA

237 maturation is completed.

238 These observations add further support to our prior suggestion (Fig. 5b) that removal of

239 the extra 33 nucleotides during normal maturation of the 3’-terminus of 16S rRNA in a wild type

240 cell involves a combination of endonucleolytic cleavage by YbeY and exoribonuclease action.

241 Strong evidence for YbeY acting an endonuclease in 16S rRNA maturation in wild type cells

242 comes from the in vivo role requirement for its crucial His114 and Arg59 residues in the presumed

243 catalytic site. Regardless of the exact position of the YbeY incision, further processing is required

244 since YbeY cleavage generates a 3’-phosphate. Additional in vivo evidence supporting an

245 endonucleolytic role of YbeY is provided by the observation reported here that three

246 exoribonucleases - RNase II, RNase R and RNase PH - are all required to mature the 3’-terminus

247 of 16S rRNA in a ΔybeY pEra strain whereas they act redundantly in a ybeY+ strain (9). As noted

248 above, it is also possible that YbeY plays an additional non-nucleolytic role as well, such as

249 interacting with ribosomal protein S11 and then helping to recruit GTP-bound Era to the 3’-end of

250 the 16S rRNA precursor (13).

251 Whether certain RNases such as RNase III, RNase Z and RNase HI are essential differs

252 between bacteria and this is true of YbeY as well (23). Even though bacteria in which YbeY is

253 essential such as Vibrio cholerae, Pseudomonas aeruginosa and Streptococcus pneumonia also

254 possess the different exoribonucleases, they might lack the type of 16S rRNA 3’-end processing

255 capabilities collectively provided by these exoribonucleases in E. coli. YbeY could therefore

256 potentially be a target of a new class of antibiotic in such organisms.

257

258 Material and Methods

259 Bacterial growth curves. Overnight bacterial cultures were diluted between 10 to 100 fold

260 in LB, supplemented with or without antibiotics. Bacterial cultures were grown to the exponential

261 growth phase. OD600 of exponentially growing bacterial cultures were adjusted to 0.05 in 6 ml of

262 LB. The growth of bacteria was measured in one hour intervals as OD600. Bacteria were grown at

263 37°C.

264 Bacterial strains, knockout generation and plasmid construction. E. coli MC4100

265 strain background was used throughout this study. Lambda red and P1 transduction techniques

266 were used for making knockouts following instructions in (30) and Sambrook manual (31),

267 respectively. pASK-IBA3C plasmid (IBA Life Sciences) was used for constructing overexpression

268 plasmid. The overexpression strains grew identical in presence or absence of 2.5 ng/ml of inducer

269 anhydrotetracycline. All growth assays were performed in absence of the anhydrotetracycline but

270 in the presence of 5 μg/ml of chloramphenicol.

271 RNA extraction and rRNA profiling. Cells were grown to OD600 between 0.5 and 1 and

272 pelleted by centrifugation. Bacterial cell pellets were frozen quickly in liquid nitrogen. Cell pellets

273 were either stored at -800C or processed immediately for RNA extraction. For figure 3 time points,

274 samples (treated or non-treated with 400μg/ml rifampicin) were frozen instantly in liquid nitrogen

275 and stored at -800C. Before extraction of RNA, we slow thawed the bacterial culture and

276 centrifuged the cells. All RNA extractions were performed using trizol (Thermo Fisher Scientific)

277 extraction method. Trizol purified RNA were cleaned using RNeasy Mini Kit (Qiagen). Total RNA

278 were fractionated as described in reference (11).

279 cDNA synthesis and qPCR. Purified RNA were treated with TURBO DNase (Thermo

280 Fisher Scientific) for the removal of DNA contamination. 500ng of DNase-treated RNA were

281 converted to cDNA using SuperScript® IV Reverse Transcriptase and random hexamer primers

282 in 20µl reaction volume. qPCR reaction mixture was prepared by combining PCR master mix

283 (LightCycler ® 480 SYBR Green I Master, Roche), gene specific primers, and 1μl of ten times

284 diluted cDNA. Thermal conditions were 5 min at 950C, followed by 40 cycles of 10sec at 950C,

285 10 sec at 530C and 30sec at 720C. Gene expression change was calculated using 2(-ΔΔCt) method.

286 Data was normalized using rpoB gene expression level as internal control. Error bars represent the

287 standard error of mean (SEM) of three biological replicates.

288 Ribosome profile. Ribosome profile was done with slight modifications as described in

289 (11). Briefly, cells were harvested from exponentially growing bacterial cultures and froze

290 immediately in liquid nitrogen. Bacterial cell pellets were then dissolved in buffer A, containing

291 lysozyme at a concentration of 1mg/ml, and incubated on ice for 30 minutes, followed by two

292 rounds of freeze-thaw cycle. Sodium deoxycholate at a concentration of 0.3% was added to the

293 bacterial suspension and incubated further on ice for 5 min. Cells debris were removed by spinning

294 lysed bacterial culture at 18,000g for 30 min. Equivalent amount of bacterial lysates were

295 fractionated in 10-40% sucrose gradient. OD of each fraction was measured at 260nm wavelength.

296 Acknowledgments

297 This work was supported by National Institutes of Health grant GM31030 to G.C.W and P30

298 ES002109 (to the MIT Center for Environmental Health Sciences). G.C.W. is an American

299 Cancer Society Professor.

300

301 Figure legends

302 Fig 1. Overproduction of Era improves the 16S rRNA processing defect in ΔybeY strain but no

303 recovery under different stress conditions. (a) qPCR analysis of mRNA showing that ΔybeY pEra

304 strain expresses 15 times more era mRNA than MC4100 pCon strain. (b) Growth curves

305 performed in LB at 37°C show that ΔybeY pEra strain grows significantly better than ΔybeY pCon

306 strain. (c) ΔybeY pEra strain has a higher amount of 16S rRNA than ΔybeY pCon strain. (d)

307 Ribosome profile shows the reduction of the amount of unassembled 30S and 50S ribosomal sub-

308 units and an increase of the amount of 70S ribosomes. (e) Spotting assay showing the growth of

309 bacterial strains under different stress conditions. Serially diluted bacterial cultures were spotted

310 on agar plates, which were incubated overnight at 370C other than the 450C plate. Bacterial cultures

311 were spotted in 10 fold serial dilutions.

312 Fig 2. Reduced GTPase activity makes Era less effective in suppressing the growth of ΔybeY strain.

313 (a) The structure of E. coli Era modeled based on the crystal structure of A. aeolicus Era (3IEV).

314 The inset shows the P17 amino acid residue (purple spheres), GDP (red) and Magnesium ion

315 (Blue). (b) Growth was performed in LB at 37°C show that the ΔybeY(peraP17R) strain grows

316 slower than the ΔybeY pEra strain.

317 Fig 3. Stability of 16S rRNA remains unaltered in ΔybeY pEra strain. Total RNA was extracted

318 from rifampicin treated or untreated bacterial cells that were incubated at 370C for indicated time

319 periods. In absence of rifampicin (first four lanes from the left), 16S rRNA is more abundant in

320 ΔybeY pEra strain in comparison to the ΔybeY pCon strain, while, in the presence of rifampicin

321 (first four lanes from the right), no strong difference was observed in the amount of 16S rRNA

322 between ΔybeY pCon strain and ΔybeY pEra strain.

323

324 Fig 4. Era-mediated rescue of defective 16S rRNA processing requires the participation of

325 exoribonuclease RNases II, RNases R and RNases PH. Growth of the double knockout mutants

326 namely ΔybeYΔrnb (a), ΔybeYΔrnr (b), ΔybeYΔrph (c) and ΔybeYΔpnp (d) containing either

327 control plasmid or recombinant plasmid bearing era gene were performed in LB at 37°C. RNA

328 gels on the right in each panel show rRNA profiles.

329 Fig 5. Models for 3’-end processing of 16S rRNA. Model (a) shows 3’-end processing of 16S

330 rRNA in a strain that overproduces Era and lacks YbeY. Model (b) highlights YbeY’s role in the

331 3’-end processing of 16S rRNA together with the exoribonucleases in a wild type strain.

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