SUPPLEMENTARY MATERIAL Supplementary

10 downloads 0 Views 6MB Size Report
5'-GTA GAG TTG CCC CTA CTC CGG TTT TCG AGA CCG ..... CCG GGC ATG GGC AGC CCA TTT CGT TTC TAC CAT AC and AtP1/2/3rev (5'-AGG. CAT GCG ...
-1-

SUPPLEMENTARY MATERIAL Supplementary Tables Table S1. Plasmids used in this study pDG148, pDG148(S/X)

expression vector containing the spac promoter (Stragier et al, 1988; Gößringer and Hartmann, 2007); pBR322-derivative, 18 copies per E. coli genome (Covarrubias et al., 1981)

pACYC177 E. coli rnpBwt

plasmid encoding E. coli rnpB (Wegscheid and Hartmann, 2006); pACYC177-derivative, ~20 copies per E. coli chromosome (Chang and Cohen, 1978)

pAtPRORP1, pAtPRORP2,

pDG148(S/X)-based

expression

vectors

for

A.

pAtPRORP3

thaliana PRORP1, 2 and 3; the encoded PRORP1 lacked the mitochondrial targeting sequence (this study; see Fig. S1A)

pAtPRORP1-His,

pAtPRORP2-

His, pAtPRORP3-His

pDG148- or pDG148(S/X)-based expression vector for recombinant A. thaliana PRORP1, 2 and 3 carrying a C-terminal His-tag (this study; see Fig. S1A), used for in vitro processing assays and Western blot analysis pUC19 vector encoding precursor tRNAHis under

pEc595

control of the T7 promoter (Sun et al, 2006)

Table S2. Bacterial strains Strain

Relevant Genotype

E. coli MG1655

E. coli K12[F-lambda-ilvG-rfb-50 rph-1] Guyer et al., 1981.

E. coli BW

E. coli MG1655[pBAD::rnpB, CamR]

Wegscheid and Hartmann, 2006

B. subtilis d7

B. subtilis YB886[Pxyl::rnpA, CamR]

Gößringer et al., 2006

B. subtilis SSB318 B. subtilis W169[Pspac::rnpB, MLSR]

Reference

Wegscheid et al., 2006

-2-

Table S3. Mapping statistics for second RNA-seq experiment with three biological replicate libraries for each strain, BW[pEcrnpB] (libraries M1-a, -b, -c), BW[pAtPRORP1] (libraries P1-a, -b, -c) and BW[pAtPRORP3] (libraries P3-a, -b, -c). Library

Reads

Mapped

tRNAs

rRNA

M1-a

12,307,745

6,537,616

134,558

19,324

M1-b

11,376,541

5,463,599

140,898

10,651

M1-c

11,922,918

5,821,077

145,544

9,554

P1-a

10,980,134

4,231,039

119,682

1,329

P1-b

12,494,519

4,939,838

144,516

3,400

P1-c

10,952,640

4,131,953

133,688

1,424

P3-a

18,288,958

2,217,474

111,464

2,195

P3-b

13,366,173

1,554,108

65,927

1,990

P3-c

12,988,132

2,802,326

141,586

10,559

Mapped reads ≥ 10 nt

-3-

Table S4. Oligonucleotides used for the production of Northern blot probes probe specific for

forward

reverse

rnpB

5’-TAA TAC GAC TCA CTA 5’-GAA GCT GAC CAG ACA TAG GAC AGT CAT TCATCT GTC GCC GCT TC AGG CCA GC

rrfH

5’-TAA TAC GAC TCA CTA 5’-TGC CTG GCG GCC GTA TAG GCA GTT CCC TAC TCT GCG CGG TGG TC CGC ATG GGGAG

tRNAAsp(GUC)

5’-ATT AAT ACG ACT CAC 5’-GGA GCG GTA GTT CAG TAT AGG CGG AAC GGA TCG GTT AGA ATA CCT GC CGG GAC TCG AAC CCG

tRNAHis(GUG)

5’-TAA TAC GAC TCA CTA 5’-GTG GCT ATA GCT CAG TTG TAG GGG TGG CTA ATG GTA G GGA TTC

tRNAIle(GAU)

5’-ATT AAT ACG ACT CAC 5’-AGG CTT GTA GCT CAG TAT AGG TAG GCC TGA GTG GTG GTT AGA GC GAC TTG AAC CAC C

tRNASec(UCA)

5’-ATT AAT ACG ACT CAC 5’-GAT CGT CGT CTC CGG TGA TAT AGG CGG AAG ATC GGC GGC ACA GGA GTC GAA CCT GC

Nucleotides in italics: T7 promoter sequence probe specific for

5'- and 3'- digoxigenin-modified DNA

tRNAArg(CCU)

5’-GCA ATT AGC CCT TAG GAG GGG CTC GT

tRNASer(CGA)

5’-GTA GAG TTG CCC CTA CTC CGG TTT TCG AGA CCG

-4-

Table S5. Generation times (G) of the E. coli BW complementation strains based on four independent experiments of the type shown in Fig. 1 of the main manuscript. G [min] 28°C

37°C

BW[EcrnpB]

BW[AtPRORP1] BW[AtPRORP2] BW[AtPRORP3]

(1)

49.1

62.1

74.5

56.4

(2)

58.3

27.2

68.9

57.8

(3)

56.8

122.3

68.7

54.0

(4)

50.9

116.9

72.2

55.8



53.8 ± 2.2

82.1 ± 22.8

71.1 ± 1.4

56.0 ± 0.8

(1)

24.0

39.0

n.d.

27.3

(2)

28.3

47.1

n.d.

32.1

(3)

29.3

54.7

n.d.

35.7

(4)

27.0

50.7

n.d.

27.4



27.1 ± 1.1

47.9 ± 3.3

n.d.

30.6 ± 2.0

n.d., not determinable Growth rate(µ): µ =

!"#$ ! !!"#$ ! ! ! !! !

Generation time (G): 𝐺=

𝑙𝑛2 µμ

OD(1) = OD600 at the beginning of the exponential growth phase. OD(2) = OD600 at the end of the exponential growth phase. t(1) = incubation time of the culture at the beginning of the exponential growth phase. t(2) = incubation time of the culture at the end of the exponential growth phase

-5-

Table S6. Cleavage site selection of RNase P enzymes in E. coli BW. Only tRNA reads with the terminal 5´-end at position -2, -1 or +1 were included in the evaluation. Nucleotide identities at the cleavage site (position -2, -1, +1 and +73) and numbers of reads starting with the terminal 5´-end at the respective position are shown. Extended AU- and GC-basepairing (between N-1/N+73 or N-2/C+74) at the acceptor stem is shown by bold red and orange nucleotides, respectively. Only tRNA species with a minimal number of 10 reads (sum of reads corresponding to 5'-ends at -2, -1 and +1) in at least two of the biological replicates were included in the evaluation. read number M1 Nr

tRNA

-2

-1

+1

+73

-2

-1

P1 +1

-2

-1

0

273

0

38

P3 +1

-2

-1

0

1

0

2

+1

1

Ala_GGC 1/2

G

U

G

A

1

1

2

Ala_GGC 2/2

A

C

G

A

1

5

3

Ala_UGC*

A

U

G

A

7

9

4532

3

879

2962

2

8

1670

4

Arg_ACG 1/2

U

U

G

A

1045

16

12

67

9

43

196

4

15

5

Arg_ACG 2/2*

A

U

G

A

442

25

36

42

28

129

247

10

44

6

Arg_CCG

A

A

G

G

NaN

NaN

NaN

2

0

29

NaN

NaN

NaN

7

Arg_CCU

U

U

G

A

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

8

Arg_UCU

A

C

G

G

NaN

NaN

NaN

0

34

5

NaN

NaN

NaN

9

Asn_GUU*

A

U

U

G

4

20

493

1

79

454

2

4

161

10

Asp_GUC 1/2*

G

U

G

G

5

10

26

327

6

5

11

Asp_GUC 2/2

G

C

G

G

2

6

2

2161

1

39

12

Cys_GCA

U

U

G

U

1

0

69

0

0

104

0

0

19

13

Gln_CUG*

G

U

U

G

5

2

300

1

102

162

0

0

82

14

Gln_UUG 1/2

G

A

U

G

0

1

0

7

0

2

15

Gln_UUG 2/2

A

U

U

G

7

2

1

19

3

2

16

Glu_UUC 1/2*

A

C

G

G

2

0

0

21

0

0

17

Glu_UUC 2/2*

G

U

G

G

0

1

0

6

0

0

18

Gly_CCC

A

A

G

U

2

3

2

6

0

17

19

Gly_GCC 1/3*

A

A

G

U

0

0

0

29

0

77

20

Gly_GCC 2/3

A

C

G

U

0

0

0

3

0

0

21

Gly_GCC 3/3

A

U

G

U

0

6

0

18

0

2

22

Gly_UCC

A

U

G

U

8

0

72

1

0

59

1

0

23

23

His_GUG

U

G

G

C

49

1082

8

3

636

736

15

349

28

24

Ile1_GAU*

A

C

A

A

7

2

337

2

65

461

1

1

97

25

Ile2_CAU 1/2

A

U

G

A

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

26

Ile2_CAU 2/2

A

A

G

A

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

27

Leu_CAA

G

U

G

A

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

28

Leu_CAG 1/3*

A

U

G

A

69

29

Leu_CAG 3/3

G

U

G

A

1

2

1165

559

322

1

426

30

Leu_CAG 2/3

G

G

G

A

0

0

1128

118

394

0

376

441

3225

533 117 636

1360

38 1 0

448

701

727 82 635

1141

15 0 0

194

1005

254 46 223

445

-631

Leu_GAG

G

U

G

A

0

0

46

0

19

28

1

0

11

32

Leu_UAA

G

A

G

A

1

1

26

1

4

473

0

0

74

33

Leu_UAG

U

U

G

A

0

0

351

0

63

288

0

0

102

34

Lys_UUU 1/5*

U

C

G

A

3

35

Lys_UUU 4/5

C

C

G

A

4

36

Lys_UUU 2/5

A

U

G

A

0

37

Lys_UUU 5/5

G

U

G

A

0

38

Lys_UUU 3/5

A

G

G

A

0

0

39

Met_CAU 1/2

A

U

G

A

NaN

NaN

40

Met_CAU 2/2

C

U

G

A

NaN

41

Phe_GAA 1/2

U

U

G

A

42

Phe_GAA 2/2

U

U

G

43

Pro_CGG

U

U

44

Pro_GGG

G

45

Pro_UGG

46

1

0

1

0 396

0 0

0

19

378

0

2

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

A

NaN

NaN

C

A

0

U

C

A

U

U

C

Sec_UCA

G

C

47

Ser_CGA

A

48

Ser_GCU

49

0 797

0 0

0

0

141

0

0

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

0

12

1

2

8

NaN

NaN

NaN

1

0

17

1

12

5

NaN

NaN

NaN

A

0

0

47

0

2

38

0

1

22

G

G

0

0

22

NaN

NaN

NaN

NaN

NaN

NaN

C

G

G

123

17

116

14

444

47

71

187

64

G

U

G

G

1

2

1132

0

19

864

1

0

384

Ser_GGA*

U

U

G

G

1

3

999

2

7

615

0

2

308

50

Ser_UGA

C

C

G

G

NaN

NaN

NaN

2

3

10

NaN

NaN

NaN

51

Thr_CGU 1/2

U

U

G

A

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

52

Thr_CGU 2/2

G

G

G

A

0

0

73

0

18

49

0

0

15

53

Thr_GGU 1/2

G

U

G

A

1

1

111

1

22

72

1

0

23

54

Thr_GGU 2/2

G

U

G

A

0

1

449

2

197

130

0

0

94

55

Thr_UGU

A

U

G

A

1

0

116

0

19

95

0

0

32

56

Trp_CCA

U

U

A

G

23

4

59

10

6

219

3

1

54

57

Tyr_GUA 1/3

C

C

G

A

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

NaN

58

Tyr_GUA 2/3

G

U

G

A

NaN

NaN

NaN

0

7

6

NaN

NaN

NaN

59

Tyr_GUA 3/3

C

U

G

A

NaN

NaN

NaN

0

7

6

NaN

NaN

NaN

60

Val_GAC 1/2

C

U

G

A

2

0

50

0

29

48

0

0

14

61

Val_GAC 2/2

U

U

G

A

0

0

363

0

122

464

0

21

119

62

Val_UAC 1/3*

G

U

G

A

1

4

1

1025

1

4

63

Val_UAC 2/3

G

G

G

A

0

1

0

88

0

0

64

Val_UAC 3/3*

U

C

G

A

3

5

2

79

0

1

65

fMet_1/3*

G

A

C

A

66

fMet_3/3

G

A

C

A

0

3

944

0

8

854

1

1

227

67

fMet_2/3

G

G

C

A

6

2

651

2

51

761

1

0

203

68

tmRNA

U

U

G

A

2

1

578

2

338

1167

0

29

638

885

735

349

NaN: not a number (≤ 10 reads in at least two biological replicates); *, two or more tRNAs combined with identical sequences between positions -2 to A76 according to the files "List of sequence merges and renaming in the above data" and "Original classification of tRNAs, including 25 nt upstream (.docx)" deposited at http://bioinf.pharmazie.unimarburg.de/supplements/prorp_2017/

-7-

Table S7. Low cleavage extent (% of processed substrate) of pre-tRNASec compared with pre-tRNAGly and pre-tRNAHis in reactions catalyzed by AtPRORP1, 2 and 3 using E. coli RNase P (EcRNase P) as control. EcRNase P

AtPRORP1*

t. o. i.

AtPRORP2

AtPRORP3

20 min

pre-tRNAGly

98.2 %

99.1 %

98.8 %

98.9 %

pre-tRNAHis

86.7 %

88.5 %

83.7 %

84.7 %

t. o. i.

15 min

pre-tRNAGly

98.6 %

98.6 %

99.2 %

98.4 %

pre-tRNAHis

88.7 %

86.6 %

85.8 %

89.5 %

t. o. i.

20 min

pre-tRNAGly

98.8 %

99.0 %

99.1 %

98.1 %

pre-tRNASec

94.7 %

22.1 %

9.4 %

11.8 %

t. o. i.

8 min

pre-tRNAGly

98.7 %

95.2 %

96.6 %

98.6 %

pre-tRNASec

79.8 %

18.7 %

7.8 %

5.1 %

15 min

t. o. i.

15 min

pre-tRNAGly

98.6 %

99.2 %

99.1 %

97.7 %

pre-tRNASec

94.7 %

28.6 %

9.5 %

8.6 %

* sum of cleavage at (-2/-1) and (-1/+1); t. o. i., time of incubation of cleavage assay; for details on the cleavage assays, see "In vitro RNase P cleavage assay" below.

-8-

Supplementary Methods Cell growth LB media (10 g/l peptone, 5 g/l yeast extract, 10 g/l NaCl, pH 7.6) for the growth of plasmidcontaining BW strains were supplemented with 25 µg/ml chloramphenicol and 100 µg/ml ampicillin. For induction or repression of chromosomal rnpB expression, 10 mM arabinose or 10 mM glucose were added to the LB medium, respectively. Cells were grown at 200 rpm at 28°C or 37°C as indicated and harvested by centrifugation for 5 min at 4,200 g and room temperature. Generally, 3 ml of a BW strain overnight culture grown in the presence of arabinose was used to inoculate 30 ml fresh arabinose-containing LB medium (starting OD600 = 0.1) to initiate a new exponential growth. At an OD600 of 0.6 – 0.8 cells were washed twice with LB medium to remove the arabinose and used to inoculate a fresh culture (70 ml, starting OD600 = 0.02) containing glucose as the carbon source (or arabinose in the case of reference cultures). Cells were grown for different time intervals before aliquots were withdrawn for OD600 measurement (growth curves), RNA preparation (Illumina sequencing or primer extension) or protein preparation (Western blot analysis). For the preparation of stationary phase cells or outgrowth phase cells (cells that have entered a new exponential phase after stationary growth) 3 ml of the respective E. coli strain was grown in the presence of arabinose for 6 h. After two washing steps with LB medium this culture was used to inoculate 50 ml fresh glucose-containing medium (OD600 = 0.01). After 14 hours growth at 200 rpm at 28°C or 37°C the stationary phase cells were used to inoculate the outgrowth culture (250 ml fresh glucose- or arabinose-containing medium, starting OD600 = 0.1). Cells of the outgrowth cultures were grown at 200 rpm at 28°C or 37°C and harvested for subsequent RNA or protein preparation at an OD600 = 1.0. Plasmid preparations for in vivo studies For in vivo complementation studies, the organellar targeting sequence was deleted from AtPRORP1 (Fig S1A). Arabidopsis thaliana PRORP cDNAs were PCR-amplified from expression vectors pET28-At_PRORP1-His, pET28-At_PRORP2-His (Pavlova et al., 2012) and pCB619-At_PRORP3 (Weber et al., 2014) using the primer pairs AtP1forw (5’-AGC CCG GGC ATG GGC AGC CCA TTT CGT TTC TAC CAT AC and AtP1/2/3rev (5’-AGG CAT GCG GAT CCT TTC AGC AAA AAA CCC CTC AAG ACC CG) for AtPRORP1 (At2g32230), AtP2forw (5’-AGC CCG GGC ATG GCT GCT TCT GAT CAA CAC C) and AtP1/2/3rev for AtPRORP2 (At2g16650) and AtP3forw (5’-AGC CCG GGC ATG GCT GGT ACT GAT AAC C) and AtP3rev (5’-AGG AAT TCG CAT GCC TGC ATC TAT GAA

-9-

CTC TGC) for AtPRORP3 (At4g21900). Restriction sites SmaI and SphI used for cloning are underlined. AtPRORP1 and AtPRORP2 contained a C-terminal 6 x His-tag sequence and a T7 terminator sequence encoded in vector pET28. The PCR fragments were inserted into the expression vector pDG148(S/X) (Gößringer and Hartmann, 2007; S/X: additional unique sites for SmaI (S) and XhoI (X) introduced between the HindIII and SphI sites downstream of the Pspac promoter of pDG148) using the restriction enzymes SmaI and SphI. The resulting expression plasmids were named pAtPRORP1-His, pAtPRORP2-His and pAtPRORP3, respectively. The plasmids pAtPRORP1-His and pAtPRORP2-His were used as templates for the construction of the respective expression vectors lacking a C-terminal 6 x His-tag sequence. This was achieved in a three-step PCR using Phusion Polymerase (Finnzymes). For the construction of vector pAtPRORP1, primers AtP1forw (see above) and AtP1rev(-) (5’-TCA CTC GAG AGG TGT TTT GGA TCT TTT TGC GC) gave rise to the first PCR product, and primers Ter7P1forw (5’-GAT CCA AAA CAC CTC TCG AGT GAG ATC CGG CTG CTA ACA AAG CCC GAA AGG) and Ter7P1rev (5-CTT GCA TGC GGA TCC TTT CAG CAA AAA ACC CCT CAA G) were employed to generate the second PCR fragment. The two fragments were combined, extended by PCR via overlapping sequences and finally amplified with the external primers AtP1forw and Ter7P1rev. Primer-encoded restriction sites (underlined) enabled the insertion into vector pDG148(S/X) via SmaI and SphI. pAtPRORP2 was constructed in a similar manner, using AtP2forw and AtP2rev(-) (5’-TCA CTC GAG AGG AAT CTT CCC ATT ACT CTT AGG) for the first PCR, primers Ter7P2forw (5’-GGG AAG ATT CCT CTC GAG TGA GAT CCG GCT GCT AAC AAA GCC CGA AAG G) and Ter7P2rev (5’-CTT GCA TGC GGA TCC TTT CAG CAA AAA ACC CC) for the second PCR, and primers AtP2forw and Ter7P2rev for the third PCR. Further steps were performed as for the construction of pAtPRORP1 (see above). The resulting expression plasmids were named pAtPRORP1 and pAtPRORP2, respectively. The plasmid pET28-At_PRORP3-His (Pavlova et al., 2012) was used as template for the construction of the expression vector pAtPRORP3-His containing a C-terminal 6 x His-tag sequence. The AtPRORP3-His gene was amplified by PCR using the primer pair AtP3cHisforw (5’-GAT CTA GAT GGC TGG TAC TGA TAA CCG CCG C) and AtP1/2/3rev. The PCR fragment was inserted into the expression vector pDG148 (Stragier et al., 1988) using the restriction enzymes XbaI and SphI. The resulting expression plasmid was named pAtPRORP3-His.

- 10 -

The PRORP2 cDNA from Trypanosoma brucei was PCR amplified from cloning vector pET21-TbPRORP2-His (Taschner et al., 2012) using the primer pairs T2forw 5’-AGT CTAGAC ATA TGC GCT CCG TGT TG (encoding an XbaI site) and T2rev 5’-AG A GGC CT CCTT TCA GCA AAA AAC C (encoding a StuI site). The PCR fragment was cleaved with XbaI and StuI for insertion into the expression vector pDG148 via the restriction enzymes XbaI and SwaI. Initial RNA-Seq analysis (see Fig. 2 of the main manuscript) For the initial RNA-Seq analysis overnight cultures of MG1655 and BW[AtPRORP1-His] were grown at 37°C/200 rpm in LB medium supplemented with 10 mM arabinose. The overnight cultures were used to inoculate 30 ml arabinose-containing fresh LB medium (starting OD600 = 0.1) to initiate exponential growth. At an OD600 ~ 0.7, cells were washed twice with LB medium to remove arabinose (5 min at 3,000 g and resuspension in 40 ml fresh prewarmed LB medium). Thereafter, the cell pellet was resuspended in 200 ml LB medium containing 10 mM glucose (starting OD600 = 0.2) and cells were grown to an OD600 of ~1.0 (~70 min). Preparation of total RNA was performed by the hot phenol method. In detail, the cell pellet of a 10–30 ml culture was resuspended in 4 ml icecold extraction buffer (10 mM NaOAc, 150 mM sucrose, 1% SDS, pH 4.8) and mixed with 1 volume 65 °C hot phenol (water-saturated, pH 4.5 – 5.0). After incubation for 5 min at 65 °C (with shaking and vigorous mixing every min) and 5 min on ice the solution was centrifuged for 20 min at 9,000 g. The phenol extraction was repeated with the aqueous upper phase at room temperature, followed by chloroform extraction and isopropanol precipitation. The RNA preparation used for Illumina sequencing was additionally treated with DNase I. The solubilized RNAs were first poly(A)-tailed using poly(A) polymerase. Then an RNA adapter was ligated to the 5’phosphate of the RNA. First- strand cDNA synthesis was performed using an oligo(dT)adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCRamplified to about 20-30 ng/µl using a high fidelity DNA polymerase. The primers used for PCR amplification were designed for amplicon sequencing according to the instructions of Illumina/Solexa. The following adapter sequences flank the cDNA inserts: 5’- end (53 bases): 5’-AATGATACGG CGACCACCGA CAGGTTCAGA GTTCTACAGT CCGACGATCN NNN-3’ (NNNN: barcode) 3’-end (45 bases): 5’-CAAGCAGAAG ACGGCATACG ATTTTTTTTT TTTTTTTTTT TTTTT-3’

- 11 -

The combined length of the flanking sequences is 98 bases. The PCR products were analyzed by capillary electrophoresis. The cDNA was purified using the Macherey & Nagel NucleoSpin Exctract II kit. Sequencing was performed on an Illumina Genome Analyzer IIx. Primer extension analysis For details on cell growth, see paragraph “Cell growth” above. Primer extension analysis was performed with 20 - 30 µg total cellular RNA prepared by the hot phenol method as described above ("First RNA-Seq analysis"). The 5'-32P-endlabeled primers were specific to mature E. coli 4.5S RNA (5'-GGA GAA CCA ACA GAG CCC-3'; annealing to nt 20 to 3 of mature 4.5S RNA), tRNAAsp(GUC) (5'-TAA CCG ACT GAA CTA CCG C-3', annealing to nt 22 to 4 of mature tRNAAsp) or tRNASer(CGA) (5'-TCA GCC GCT CCG GCA TCT C-3', annealing to nt 22 to 4 of mature tRNASer). The primers were annealed to the total RNA in 10 µl 1 x cDNA synthesis buffer (ThermoScript, ThermoScientific Invitrogen) for 60 min applying a linear temperature gradient from 90 to 55 °C. Reverse transcription was performed by adding 5 µl RT-mix (1 x cDNA synthesis buffer containing 4.5 units ThermoScript reverse transcriptase, 15 mM DTT and 3 mM dNTP mix) and incubation for 50 min at 55 °C (E. coli 4.5S RNA, tRNAAsp) or 65°C (tRNASer). The primer extension products were analyzed by 20% PAGE in the presence of 8 M urea. As size markers we used 5´-32P-endlabeled DNA primers: (i) in the case of 4.5S RNA, the 4.5S RNA-specific primer (see above) 3’-extended by 26 nt (5’-GGA GAA CCA ACA GAG CCC CCA TTG AGA GCG TTG AGA ACC AAC GC) or 42 nt (5’-GGA GAA CCA ACA GAG CCC CCA TTG AGA GCG TTG AGA ACC AAC GGG GAA TTC CGG TCT CCC), respectively, complementary to the 5’-region of the ffs gene (encoding 4.5S RNA); (ii) the tRNAAsp(GUC)-specific primer (see above) 3’extended by 3 nt (5'-TAA CCG ACT GAA CTA CCG CTC C-3'; corresponding to cleavage at nt -1/+1) or 5 nt (5'-TAA CCG ACT GAA CTA CCG CTC CGC-3'; corresponding to cleavage at nt -3/-2), respectively, complementary to the 5’-region of the aspV gene (encoding tRNAAsp(GUC)); (iii) the tRNASer(CGA)-specific primer (see above) 3’-extended by 3 nt (5'TCA GCC GCT CCG GCA TCT CTC C-3'; corresponding to cleavage at nt -1/+1) or 4 nt (5'TCA GCC GCT CCG GCA TCT CTC CG-3'; corresponding to cleavage at nt -2/-1), respectively, complementary to the 5’-region of the serU gene (encoding tRNASer (CGA)). Northern blot analysis For Northern blots, cells to be analyzed were grown overnight to stationary phase in the presence of 10 mM glucose. Overnight cultures were diluted with fresh glucose-containing LB medium to a starting OD600 of 0.2 and grown in a new exponential phase (termed outgrowth from stationary phase) to an OD600 of 1.0, followed by cell harvest (centrifugation

- 12 -

for 5 min at 4,200 g and room temperature). Northern blots were basically done as described in Li et al. (2011), with minor modifications. Total cellular RNA for Northern blot analysis of tRNAs was prepared according to Method 1 (“Extracting RNA three times with hot phenol.”) described by Damm et al. (2015). Total RNA (2 - 12 µg per lane) was separated on 8 to 10% polyacrylamide gels containing 8 M urea. Chromosomal E. coli BW DNA was used to amplify the 5S RNA gene (rrfH), and chromosomal DNA of E. coli MG1655 was used to amplify the genes encoding RNase P RNA (rnpB), tRNASec, tRNAAsp, tRNAHis and tRNAIle (see Table S3 for primer sequences). The 5S RNA served as internal control for transfer efficiency of RNA onto the nylon membrane and the quality of the total RNA preparations. The respective forward primers encoded a T7 promoter sequence (nucleotides in italics in Table S3) that enabled the synthesis of antisense transcripts. For the production of such internally labeled antisense probes, T7 transcription was performed in the presence of digoxigenin-11-UTP (Roche Diagnostics). For the detection of tRNAAsp, tRNAHis and tRNAIle, antisense transcripts complementary to the full-length tRNA were prepared. For the detection of tRNAArg and tRNASer, isoacceptor-specific DNA probes containing dual digoxigenin labels at the 5'- and 3'-end (Eurofins Genomics) were used. For detection of E. coli RNase P RNA, the nylon membrane was simultaneously hybridized with the rnpB- and rrfH-specific probes. For analysis of tRNASec levels the nylon membrane was first hybridized with the tRNASec-specific probe, followed by a stripping step (see below) and then hybridized with the rrfH-specific probe. Stripping was performed twice for 60 min at 80°C with stripping solution (50% deionized formamide; 5% SDS; 50 mM Tris-HCl, pH 7.5), followed by rinsing the membrane thoroughly with ddH2O and reprobing with the rrfHspecific probe. Western blot analysis B. subtilis or E. coli cells harboring complementation plasmids were grown in 50 ml LB broth in the presence of inducer (B. subtilis d7: 2 % xylose / B. subtilis SSB318: 1 mM IPTG / E. coli BW: 10 mM arabinose) to an OD600 ≈ 0.9, and harvested by centrifugation for 5 min at 5,000 g. After resuspension in 3.5 ml lysis buffer (50 mM Tris, pH 8.0; 100 mM NaCl; 0.1 % Triton X-100; 1 M NH4Cl) supplemented with protease inhibitors (Complete Mini, EDTAfree, Roche) and lysozyme (300 µg/ml final concentration), cells were incubated for 15 min on ice. After sonication for 12 min (Branson Sonifier: duty cycle 40%; output 3) in an icewater bath, cell debris was removed by centrifugation for 7 min at 150 g/ 4°C. The cell lysate was then transferred to a new reaction tube and centrifuged for 45 min at 13,000 g / 4°C to separate soluble and insoluble protein fractions. The volume of the supernatant (soluble

- 13 -

protein fraction) was determined, and the pellet (insoluble protein fraction) was resuspended in the same volume TUS buffer (50 mM Tris-HCl, 100 mM NaCl, 8 M urea, pH 7.4). For immunodetection of His-tagged TbPRORP2, 30 µg of total protein and 30 µg of the soluble protein sample were loaded onto a 15% SDS-PAA gel (Laemmli, 1970). Because the protein concentration of the insoluble fraction was too low to be determined, a volume identical to that of the soluble protein fraction (containing 30 µg protein) was applied to the gel. After electrophoresis, proteins were transferred onto a PVDF membrane via semi-dry blotting. After blocking with an alkali-soluble casein solution (Novagen) the membrane was incubated with a monoclonal antibody (IgG) directed against the His-tag (Novagen). For the detection of untagged AtPRORP1 (Fig. S9C), a polyclonal antiserum (Gobert et al., 2010) was used. Specific immunocomplexes were detected with secondary antibodies coupled to alkaline phosphatase (Qiagen) using the BCIP/NBT colour development substrate (Promega). For analyzing the expression levels of heterologously expressed AtPRORP1, AtPRORP2 and AtPRORP3 in E. coli BW, the plasmid-encoded AtPRORP isoenzymes carried a Cterminal His(6)-tag. Cells were grown in glucose medium as described in the paragraph “Cell growth” (see above) and harvested in the exponential growth phase (OD600 ~ 1.0) by centrifugation for 5 min at 5,000 g. After resuspension of the cells (OD600 ~ 10 ) in 600 µl lysis buffer (25 mM Tris-HCl, pH 8.0; 100 mM NaCl; 2 mM EDTA, 0.1% Triton X-100) supplemented with protease inhibitors (Complete Mini, EDTA-free, Roche) and lysozyme (300 µg/ml final concentration), cells were incubated for 15 min on ice. After cell disruption using the FastPrep System (MP Biomedicals) with pre-chilled lysing matrix B at a speed of 6.0 for 30 s, cell debris was removed by centrifugation for 7 min at 150 g / 4°C. Separation of soluble and insoluble protein fractions was performed as described above. Immunodetection of soluble and insoluble fractions of His-tagged AtPRORP1, AtPRORP2 and AtPRORP3 was carried out as described for TbPRORP2, with the exception that 20 µg of soluble protein fraction per lane were loaded onto a 12% SDS-PAA gel (Laemmli, 1970). In vitro RNase P cleavage assay The 5´-processing activity of A. thaliana PRORP isoenzymes was tested using Thermus thermophilus pre-tRNAGly (Hartmann et al., 1991; Busch et al., 2000), E. coli pre-tRNAHis (Sun et al., 2006), E. coli pre-tRNASec and E. coli 4.5S RNA. T. thermophilus pre-tRNAGly and E. coli pre-tRNAHis were synthesized by T7 transcription from linearized plasmid DNA as described previously (Busch et al., 2000; Sun et al., 2006). Templates for E. coli pretRNASec and pre-4.5S RNA were amplified using the primer pairs 5-ATT AAT ACG ACT CAC TAT AGG CAA TCG AGG CGC GGA AGA TCG TCG-3 / 5-TGG CGG AAG ATC

- 14 -

ACA GGA GTC GAA CCT GC-3 and 5-ATT AAT ACG ACT CAC TAT AGG TTG GTT CTC AAC GCT CTC AAT GGG GG-3 / 5-ATG GGT GGG GGC CCT GCC AGC TAC ATC C-3, respectively (T7 promoter sequences in the forward primers are shown in italics; precursor nucleotides are underlined). The PCR product was gel-purified and used as template for in vitro transcription. Preparation of AtPRORP1, AtPRORP2 and AtPRORP3 was performed as described (Pavlova et al., 2012). The cleavage reactions were performed at 28°C (AtPRORP2) or 37°C (AtPRORP1 and AtPRORP3) under single turnover conditions ([E]>>[S]) in the presence of 120 nM AtPRORP and trace amounts of 5´-32P-endlabeled substrate (< 1 nM) in 50 mM Tris/HCl, pH 7.0; 20 mM NaCl; 4.5 mM MgCl2; 20 µg/ml BSA; 5 mM DTT; 0.4 units/µl Ribolock RNase Inhibitor (Life Technologies). After preincubation of substrate (5 min at 55°C and 25 min at 37°C) and AtPRORP (AtPRORP1 and AtPRORP3 for 5 min at 37°C, AtPRORP2 for 5 min at 28°C) in cleavage buffer (preincubation of substrate without DTT and preincubation of the enzymes without Ribolock RNase Inhibitor), both components were combined to start the reaction. Samples were withdrawn after indicated time periods and analyzed by 20% denaturing PAGE. In parallel, reactions with the RNase P holoenzyme from Escherichia coli (100 nM RNase P RNA; 800 nM RNase P protein) were performed under the same reaction conditions. Here, the RNase P RNA was preincubated for 5 min at 55°C, 30 min at 37°C and another 5 min at 37°C in the presence of the RNase P protein before substrate was added. Samples were withdrawn after indicated time periods of incubation at 37°C and analyzed by 20% denaturing PAGE.

- 15 -

Supplementary Figures CLUSTAL 2.1 AtPRORP3

------------------------------------------------------------------MAGTDNRRSRHDD-

AtPRORP2

------------------------------------------------------------------MAASDQHRSRRHD-

AtPRORP1

MLRLTCFTPSFSRACCPLFAMMLKVPSVHLHHPRFSPFRFYHTSLLVKGTRDRRLILVERSRHLCTLPLAAAKQSAASPS

Structure

--------------------------------------------------------------------------------

:. :

::*

recomb. AtPRORP1

---------------------------------MGSPFRFYHTSLLVKGTRDRRLILVERSRHLCTLPLAAAKQSAASPS

AtPRORP3

ESPKNPNKKKKGNRNPEKSLLINLHSCSKRKDLSAALALYDAAITSSDIRLNQQHFQSLLYLCSAFISDP-SLQTVAIDR

AtPRORP2

ESSSRPNKKKKVSRNPETNLLFNLNSCSKSKDLSAALALYDAAITSSEVRLSQQHFQTLLYLCSASITDI-SLQYLAIDR

AtPRORP1

ENLSRKAKKKAIQQSPEALLKQKLDMCSKKGDVLEALRLYDEARRN-GVQLSQYHYNVLLYVCSLAEAATESSPNPGLSR *. ..

***

.:.**

*

:*. ***

*:

** *** *

.

::*.* *:: ***:**

:

*

.:.*

Structure

--------------xxxxxxA1xxxxxxx---xxxxxA2xxxxxx------xxxxxxA3xxxxx----------xxxxxx

AtPRORP3

GFQIFDRMVSSGISPNESSVTAVARLAAAKGDGDYAFKLVKDLVAVGGVSVPRLRTYAPALLCFCDTLEAEKGYEVEDHM

AtPRORP2

GFEIFDRMVSSGISPNEASVTSVARLAAAKGNGDYAFKVVKEFVSVGGVSIPRLRTYAPALLCFCEKLEAEKGYEVEEHM

AtPRORP1

GFDIFKQMIVDKVVPNEATFTNGARLAVAKDDPEMAFDMVKQMKAFG--IQPRLRSYGPALFGFCRKGDADKAYEVDAHM **:**.:*: . : ***::.*

****.**.: : **.:**:: :.*

****:*.***: ** . :*:*.***: **

Structure

A4xxxxxxxx-----xxxxxxA5xxxx----xxxxxxA6xxxxxx--------xxxxA7xxxxxxx--xxxxxA8xxxxx

AtPRORP3

-DASGIVLEEAEISALLKVSAATGRENKVYRYLQKLRECVGCVSEETSKAIEEWFYGVKASEVSDNGIGSDIELLRAAVL

AtPRORP2

-EAAGIALEEAEISALLKVSAATGRENKVYRYLHKLREYVGCVSEETLKIIEEWFCGEKAGEVGDNGIGSDVGMLREAVL

AtPRORP1

GVESEVVPEEPELAALLKVSMDTKNADKVYKTLQRLRDLVRQVSKSTFDMIEEWFKSEVATKTG--VKKWDVKKIRDAVV : :. **.*::******

* . :***: *::**: *

**:.* . ***** .

* :.. :

*:

:* **:

Structure

-xx----xxxxxA9xxxxxxxx---xxxxxA10xxxxxx-----xxxA11xxxxxx-xA12xx------xxxA13xxxxx

AtPRORP3

KNGGGWHGLGWVGEGKWIVKKGNVSSAGKCLSCDEHLACVDTNEVETEDFVNSLVTLAMERKAKMNSCEPMADFSEFQEW

AtPRORP2

NNGGGWHGHGWVGEGKWTVKKGNVSSTGRCLSCSEQLACVDTNEVETQKFVDSLVALAMDRKTKMNSCETNVVFSEFQDW

AtPRORP1

SGGGGWHGQGWLGTGKWNVKRTEMDENGVCKCCKEKLVCIDINPVETETFAASLTRLACEREVKAN-------FNQFQEW ..****** **:* *** **: ::.. * * .*.*:*.*:* * ***: *. **. ** :*:.* *

*.:**:*

Structure

x-----------------------------------------xxxxxA14xxxxxxxxxxx------------xxA15xx

AtPRORP3

LEKHGDYEAILDGANIGLYQQNFADGGFSLPQLEAVVKELYNKSGSKKQPLILLHKKRVN-ALLENPNHRNLVEEWINNN

AtPRORP2

LEKHGDYEAIVDGANIGLYQQNFVDGSFSLSQLESVMKELYRESGNNKWPLILLHKRRVK-TLLENPTHRNLVEEWISNG

AtPRORP1

LERHGPFDAVIDGANMGLVNQ----RSFSFFQLNNTVQRCQQISPSKRLPLVILHKSRVNGGPATYPKNRALLEKWKNAG **:** ::*::****:** :*

Structure

.**: **: .::.

. * .:: **::*** **:

*.:* *:*:* . .

-------------xA16x-------------xxxA17xxxxxx------------xA18x----xxxA19xxxxxxxxxx

- 16 -

AtPRORP3

VLYATPPGSNDDWYWLYAAAKLKCLLVTNDEMRDHIFELLSNSFFQKWKERHQVRFTFVKG-CLKLEMPPPFSVVIQESE

AtPRORP2

VLYATPPGSNDDWYWLYAAAKLKCLLVTNDEMRDHIFELLGSTFFQKWKERHQVRYTFVKG-NLKLEMPSPFSVVIQESE

AtPRORP1

ALYATPPGSNDDWYWLYAAVSCKCLLVTNDEMRDHLFQLLGNSFFPRWKEKHQVRISVTREDGLKLNMPPPYSIVIQESE .******************.. *************:*:**..:** :***:**** :..:

***:**.*:*:******

Structure

----------xxxA20xxxxxxx-----------xxA21x----xxxA22xx----------------------------

AtPRORP3

KGSWHVPITSQDKEESLRSWMCITRQSS-----------

AtPRORP2

KGSWHFPVSCENNEESSRTWMCISRQSILDSPKSNGKIP

AtPRORP1

DGTWHVPMSVEDDLQTSRQWLCAKRSKTP---------.*:**.*:: ::. :: * *:* .*..

Structure

---------------------------------------

recomb. AtPRORP1

WHVPMSVEDDLQTSRQWLCAKRSKTPLEHHHHHH

Fig. S1A. Clustal 2.1 alignment of Arabidopsis thaliana (At) PRORP1, 2 and 3. The secondary structural elements inferred from the X-ray structure of AtPRORP1 (Howard et al., 2012) are numbered A1–A22 in the fifth line. The amino acid positions of the pentatricopeptide repeat domain (black letters on gray shading), the central domain (white letters on black shading) and the metallonuclease domain (white letters above pink background) are indicated. NCBI Nucleotide Accession no.: AtPRORP1, NM_179855.3/ AtPRORP2, NM_127217.4 / AtPRORP3, NM_118311. Note that the AtPRORP3 initiation codon was not the one specified in databases but the second ATG of the sequence, which we think is the one that is utilized in vivo. Thus, the AtPRORP3 sequence given here lacks the N-terminal extension “MKLKKPSLPS SLLCAVPPCL SQIRLLIPRR VRVSSSTFAN AKLVTLRNHT VNLHIYYCS” provided with NM_118311. For complementation in E. coli BW cells, the mitochondrial targeting sequence “MLRLTCFTPS FSRACCPLFA MMLKVPSVHL HHPRF“ of native precursor AtPRORP1 was replaced with the dipeptide “MG” (marked in yellow). The His-tagged version of recombinant AtPRORP1 additionally carried the Cterminal extension LEHHHHHH highlighted in yellow. Symbols in the consensus line (fourth line): * (asterisk), position with a single, fully conserved residue; : (colon), conservation between amino acid (aa) groups of strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix); . (period), conservation between aa groups of weakly similar properties (scoring ≤ 0.5 in the Gonnet PAM 250 matrix). In the PPR domain (199 aa), AtPRORP1 shares 86 identical aa (43.2% sequence identity) and 35 (:) similar aa with AtPRORP2/3 based on the alignment shown here. In the NYN domain (179 aa), AtPRORP1 shares 94 idenitical aa (52.5% sequence identity) and 32 (:) similar aa with AtPRORP2/3.

- 17 -

Fig. S1B. Alignment (Clustal 2.1 multiple sequence alignment) of the three PRORP enzymes from A. thaliana. For the alignment, the N-terminal sequence stretch “MLRLTCFTPS FSRACCPLFA MMLKVPSVHL HHPRFSPFRF YHTSLLVKGT RDRRLILVER SRHLCTLP” of AtPRORP1 (see Fig. S1A) was excluded. Sequence identity is indicated by black and similarity by gray numbers. Similarity (strongly similar properties) is defined according to Clustal Omega: STA; NEQK; NHQK; NDEQ; QHRK; MILV; MILF; HY; FYW.

- 18 -

Fig. S1C. AtPRORP1-3 structure alignment. AtPRORP2 (magenta) and AtPRORP3 (blue) 3D folds were predicted with the program Phyre2 using the AtPRORP1 3D structure (green; Howard et al., 2012; PDB ID: 4G23) as template. All three structures were then aligned (Fig. S1A) using the software STAMP (Structural Alignment of Multiple Proteins). The three domains of PRORP proteins are depicted; PPR, pentatricopeptide repeat.

- 19 -

Fig. S2. (A) Northern blot analysis of RNase P RNA (P RNA) levels in E. coli MG1655 (lane 1), in E. coli BW transformed with the empty expression vector pDG148(S/X) (lane 2) or the same plasmid expressing AtPRORP1 (lane 3). After growth in arabinose-free medium for 180 min, total cellular RNA was prepared from each strain and subjected to 8% denaturing PAGE (2 µg RNA loaded per lane), followed by blotting onto a nylon membrane. E. coli P RNA was hybridized with a specific digoxigenin-labeled antisense RNA probe. Simultaneously, the 5S RNA was probed to control for RNA loading and quality. MG1655 is the parental strain of E. coli BW (Wegscheid and Hartmann, 2006). The result demonstrates that endogenous P RNA is undetectable after 3 h of growth in the absence of arabinose. (B) LB agar plates with E. coli BW cells transformed with the empty expression vector pDG148(S/X), pAtPRORP1, pAtPRORP2 and pAtPRORP3. Cells were grown for ~16 h under permissive (arabinose) or non-permissive (glucose) conditions at 37°C or 28°C. C-terminally Histagged versions of the three A. thaliana PRORP enzymes (used for in vitro processing studies, see Fig. S1A) gave identical complemention results, ruling out the possibility that a His-tag might negatively affect the function of AtPRORP1, 2 or 3 (data not shown).

- 20 -

Fig. S3. Western blot analysis of AtPRORP1, 2 and 3 expression in E. coli BW bacteria. (A) Levels of plasmid-encoded AtPRORP isoenzymes carrying a C-terminal His-tag in soluble (S) and insoluble (IS) protein fractions prepared from cell cultures grown in glucose-containing medium at 28°C or 37°C. Corresponding protein fractions from E. coli BW transformed with an expression vector for E. coli rnpB were used as control. The protein fractions (20 µg per lane) were separated by 12% SDS-PAGE and blotted onto PVDF membranes for detection with His-tag-specific monoclonal antibodies. Migration of marker proteins (M) and their sizes in kDa are indicated on the left. Putative full-length AtPRORP1 (62.1 kDa), AtPRORP2 (60.4 kDa) and AtPRORP3 (58.7 kDa) are indicated by black arrowheads. The prominent second signal below the main band in BW[AtPRORP1-His] fractions may originate from usage of a downstream GUG start codon in E. coli cells, resulting in a AtPRORP1 variant shortened by 13 aa. The analysis demonstrates that the expression levels of the three AtPRORP isoenzymes are comparable and the enzymes are predominantly in the soluble protein fraction. Some degradation products are visible in protein fractions from BW[AtPRORP1-His] and BW[AtPRORP2His]. Higher growth temperatures (37°C vs. 28°C) result in increased levels of AtPRORP1 and AtPRORP3 in the insoluble protein fraction. (B) In parallel, aliquots of the protein fractions (2 µg per lane) were separated by 12% SDS-PAGE and stained with Coomassie Blue.

- 21 CLUSTAL 2.0.10 multiple sequence alignment mature 4.5S RNA pre-4.5S RNA

------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACCC------------TAATGCGCCTGCGCGTTGGTTCTCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACCCATTTCTGCCTCCC

{ + 393 reads } ID-246_READ-1_OCC-1 ID-168_READ-1_OCC-1 ID-114_READ-2_OCC-1 ID-382_READ-1_OCC-1 ID-84_READ-1_OCC-1 ID-255_READ-1_OCC-1 ID-259_READ-1_OCC-1 ID-425_READ-1_OCC-1 ID-36_READ-1_OCC-1 ID-23_READ-1_OCC-1 ID-338_READ-1_OCC-1 ID-50_READ-3_OCC-1 ID-135_READ-1_OCC-1 ID-245_READ-1_OCC-1 ID-249_READ-2_OCC-1 ID-235_READ-1_OCC-1 ID-217_READ-1_OCC-1 ID-220_READ-2_OCC-1 ID-315_READ-1_OCC-1 ID-104_READ-1_OCC-1 ID-66_READ-1_OCC-1 ID-142_READ-10_OCC-1 ID-256_READ-7_OCC-1 ID-151_READ-1_OCC-1 ID-316_READ-1_OCC-1 ID-320_READ-3_OCC-1 ID-197_READ-1_OCC-1 ID-224_READ-1_OCC-1 ID-223_READ-1_OCC-1 ID-144_READ-1_OCC-1 ID-119_READ-5_OCC-1 ID-227_READ-3_OCC-1 ID-63_READ-1_OCC-1 ID-140_READ-1_OCC-1 ID-182_READ-1_OCC-1 ID-153_READ-2_OCC-1 ID-98_READ-1_OCC-1 ID-230_READ-4_OCC-1 ID-26_READ-3_OCC-1 ID-84_READ-4_OCC-1 ID-255_READ-1_OCC-1 ID-218_READ-1_OCC-1 ID-240_READ-1_OCC-1 ID-172_READ-2_OCC-1 ID-126_READ-1_OCC-1 ID-21_READ-1_OCC-1 ID-66_READ-1_OCC-1 ID-82_READ-3_OCC-1 ID-205_READ-1_OCC-1

-----------CGCGTTGGTTCTCAATGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCA------------------------------------------------------------------GCGTTGGTTCTCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGC--------------------------------------------------------------------------------------------------------------------CGTTGGTTCTCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGA------------------------------------------------------------------TTTGGTTCTCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGAT------------------------------------------------------------------------CTCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCACGGCAGATGACGCTT-----------------------------------------------------------------CTCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGCAGCAGCCAAGGCAGATGACGCGT-----------------------------------------------------------------CTCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGT------------------------------------------------------------------TCAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTG------------------------------------------------------------------CAACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGT------------------------------------------------------------------AACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTG------------------------------------------------------------------ACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGCAGGAAGCAGCCAAGGCAGATGACGCGTGTGC-----------------------------------------------------------------ACGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGC------------------------------------------------------------------CGCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCC------------------------------------------------------------------GCTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCG------------------------------------------------------------------CTCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGG------------------------------------------------------------------TCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGG-----------------------------------------------------------------TCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGG-----------------------------------------------------------------TCTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGG--------------------------------------------------------------------------------------CTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGCCGCGTGTGCCGGGA-----------------------------------------------------------------CTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTT----------------------------------------------------------------------------------------------------------------------CTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGG------------------------------------------------------------------CTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGA-----------------------------------------------------------------CTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGA-----------------------------------------------------------------CTCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGG-------------------------------------------------------------------TCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATG---------------------------------------------------------------------------------TCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGAT-----------------------------------------------------------------TCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGAGGCGTGTGCCGGGAT-----------------------------------------------------------------TCAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGAT------------------------------------------------------------------CAATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATG------------------------------------------------------------------AATGGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCC---------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATG--------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGG------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAG-----------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGT------------------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTAC--------------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCC------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAG-----------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTA-------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCT-------------------------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTT----------------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCCACTCTGTTT----------------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGC-------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGC----------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGCTGTAGC-----------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTC--------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCTGGGATGTAGC-----------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGAGCCGGGATGTAGC-----------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACC-------------------------------------------------------------------------------------------------------------------------GGGAGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGC------------------------------

{ + 199 reads } ID-87_READ-1_OCC-1 ID-436_READ-16_OCC-1 ID-229_READ-2_OCC-1 ID-324_READ-2_OCC-1 ID-346_READ-1_OCC-1 ID-225_READ-1_OCC-1 ID-224_READ-1_OCC-1 ID-213_READ-1_OCC-1 ID-143_READ-126_OCC-1 ID-361_READ-2_OCC-1 ID-366_READ-1_OCC-1 ID-167_READ-1_OCC-1 ID-89_READ-14_OCC-1 ID-304_READ-1_OCC-1 ID-131_READ-27_OCC-1 ID-156_READ-1_OCC-1 ID-4_READ-1_OCC-1 ID-112_READ-24_OCC-1 ID-165_READ-1_OCC-1 ID-214_READ-1_OCC-1 ID-248_READ-1_OCC-1 ID-234_READ-1_OCC-1 ID-314_READ-1_OCC-1 ID-210_READ-1_OCC-1 ID-89_READ-2_OCC-1 ID-231_READ-1_OCC-1 ID-40_READ-1_OCC-1 ID-38_READ-1_OCC-1 ID-343_READ-1_OCC-1 ID-251_READ-1_OCC-1 ID-99_READ-1_OCC-1 ID-68_READ-1_OCC-1 ID-275_READ-1_OCC-1 ID-258_READ-7_OCC-1 ID-393_READ-3_OCC-1 ID-162_READ-1_OCC-1 ID-238_READ-1_OCC-1 ID-201_READ-1_OCC-1 ID-248_READ-1_OCC-1 ID-98_READ-1_OCC-1 ID-261_READ-1_OCC-1 ID-101_READ-1_OCC-1 ID-145_READ-1_OCC-1 ID-113_READ-1_OCC-1 ID-137_READ-5_OCC-1 ID-38_READ-5_OCC-1 ID-117_READ-1_OCC-1 ID-221_READ-3_OCC-1 ID-381_READ-1_OCC-1 ID-209_READ-1_OCC-1

------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTGGC-----------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTAC--------------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGG----------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCC--------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAG--------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGT----------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGG------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTAC-AGGTCAGGTCCGG------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTT----------------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAG-----------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTG-------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAG------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGC----------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTC---------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACC-------------------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCATGTCCGGAAGGAAGCAGCCAAGG--------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCATGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATG--------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGG------------------------------------------------------------------------------------------------------------GGGGGCTCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGACGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGC-----------------------------------------------------------------------TCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGC-------------------------------------------------------------------TCTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAG------------------------------------------------------------------CTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACTCGTGTGCCGGGATGTAGCGGGCAGG-----------------------------------------------------------------CTGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGG------------------------------------------------------------------TGTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGG-----------------------------------------------------------------TTTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGG------------------------------------------------------------------TTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGTC-----------------------------------------------------------------GTTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGC------------------------------------------------------------------TTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGT------------------------------------------------------------------------------------------TTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCCGGGCC-----------------------------------------------------------------TTGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCC------------------------------------------------------------------TGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCC-----------------------------------------------------------------TGGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCC------------------------------------------------------------------GGTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCC------------------------------------------------------------------GTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCC-----------------------------------------------------------------GTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCC-----------------------------------------------------------------GTTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGTGCCCCC------------------------------------------------------------------TTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGCGCCGGGATGTAGCTGGCAGGGCCCCC------------------------------------------------------------------TTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCC------------------------------------------------------------------TTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCA-----------------------------------------------------------------TTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGCGTGCCGGGATGTAGCTGGCCGGGCCCCC------------------------------------------------------------------TTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCA-----------------------------------------------------------------TTCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAATCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCA------------------------------------------------------------------TCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAATGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCAC-----------------------------------------------------------------TCTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCAC------------------------------------------------------------------CTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACC-----------------------------------------------------------------CTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACC-----------------------------------------------------------------CTCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGCGCCGGGATGTAGCTGGCAGGGCCCCCACC------------------------------------------------------------------TCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACCC-----------------------------------------------------------------TCCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACC-------------------------------------------------------------------CCCGCAACGCTACTCTGTTTACCAGGTCAGGTCCGGAAGGAAGCAGCCAAGGCAGATGACGCGTGTGCCGGGATGTAGCTGGCAGGGCCCCCACCCA------------

{ + 2357 reads }

Fig. S4. Alignment of the different types of cDNA reads mapping to the 4.5S RNA locus, exemplarily illustrated for the MG1655 wild type strain expressing the native E. coli RNase P. Reads mapping to the 4.5S RNA locus were grouped in three categories: (i) those overlapping the mature 5’-terminus, attributable to 5’-precursor molecules (red lettering), (ii) reads starting at the known mature 5’-terminus (green lettering), and (iii) reads corresponding to internal 4.5S RNA fragments (blue lettering). For clarity reasons, 393 red, 199 green and 2357 blue reads have been omitted. For the MG1655 wild type strain, the total number of 4.5S RNA-specific reads was 3311, of which 444 (13.4%) represented precursor molecules, 460 (13.9%) 5'-mature molecules and 2407 (72.7%) internal fragments. 8621 4.5S RNA-specific reads were obtained for the BW[AtPRORP1] strain, of which 5676 reads (65.8%) corresponded to precursor molecules, 772 (9%) to mature 4.5S RNA and 2173 (25.2%) to internal fragments.

- 22 -

Fig. S5. (A) Secondary structure of precursors to E. coli tRNAIle1(GAU) (encoded by ileT, ileU and ileV) and tRNAArg(CCU) (encoded by argW). Ten nucleotides of the 5´-leader are shown, respectively. Pre-tRNAArg can form an extended acceptor stem involving an additional U-1:A72 base pair and pre-tRNAIle1 has the idiosyncratic feature of a A1:U72 base pair. The canonical cleavage sites are indicated by black arrows. (B, C) Northern blot detection of tRNAIle1(GAU) and tRNAArg(CCU), respectively. Total cellular RNA isolated from outgrowth (exponential phase) cultures (at OD600 ~ 1.0) grown in glucose medium (see Suppl. Methods “Cell growth” and “Northern blot analysis” for details). Lanes 1: E. coli MG1655 cells (wild type); lanes 2: BW[EcrnpB] (isogenetic to MG1655 with respect to the identity of RNase P); lanes 3: BW[AtPRORP1]; lanes 4: BW[AtPRORP3]. The blot was hybridized with digoxigenin-labeled probes specific to tRNAIle(GAU) and tRNAArg(CCU), respectively. The faster migrating band in panel B was reproducibly found to be enhanced in BW[AtPRORP1], but not in BW[AtPRORP3] cells (cf. lanes 3 and 4). The nature of this band is unclear. The faint band above mature tRNAArg(CCU) could be precursor molecules with 3-5 extra nucleotides at the 5'-end according to the RNA-seq data. Putative mature tRNAs are marked by black arrowheads and putative pre-tRNAArg(CCU) by the gray arrowhead.

- 23 -

Fig. S6. LB agar plates with conditional RNase P mutant strains of B. subtilis. The mutant strains were transformed with empty vector pDG148(S/X), pAtPRORP1 and pAtmPRORP1 and grown for ~16 h under permissive or non-permissive conditions at 37°C. AtmPRORP1 is an enzymatically inactive AtPRORP1 that contains two mutations (D474A/D475A) in the active site (Gobert et al., 2010) (A) B. subtilis d7 expresses the RNase P protein under control of a xylose-inducible promoter (Gößringer et al., 2006; Gößringer and Hartmann, 2007). Replacing xylose with glucose effectively inhibits expression of the RNase P protein subunit. (B) In B. subtilis SSB318, sufficient expression of endogenous RNase P RNA depends on the presence of IPTG (Wegscheid et al., 2006).

- 24 -

Fig. S7. Westernblot analysis of AtPRORP1 expression in B. subtilis strains (A) d7 and (B) SSB318. Expression of plasmid-encoded AtPRORP1 and enzymatically inactive AtmPRORP1 was analyzed in total protein extracts (T), as well as insoluble (IS) and soluble (S) protein fractions thereof, from strains d7[AtPRORP1], d7[AtmPRORP1], SSB318[AtPRORP1] and SSB318[AtmPRORP1], respectively. As control, B. subtilis strains harboring the empty vector (d7[vector] and SSB318[vector]) were analyzed as well. The protein fractions (25 µg per lane) were separated by 15% SDS-PAGE and blotted onto PVDF membranes for detection with an AtPRORP1-specific antiserum. Migration of marker proteins (M) and their sizes in kDa are indicated on the left. Putative truncated (open arrow) variants of AtPRORP1 and AtmPRORP1 are indicated.

- 25 -

Fig. S8. LB agar plates with conditional RNase P mutant strains of B. subtilis and E. coli. The strains either contained (as control) the empty vector pDG148(S/X) or the expression vector pTbPRORP2 encoding Trypanosoma brucei PRORP2 (Taschner et al., 2012). (A) The B. subtilis d7 strain expresses the chromosomal RNase P protein under control of a xylose-inducible promoter. Replacing xylose (xyl) with glucose (glu) effectively inhibits expression of the RNase P protein subunit. (B) In the B. subtilis strain SSB318, expression of the endogeneous RNase P RNA depends on IPTG. (C) Expression of endogenous RNase P RNA in E. coli BW requires arabinose (ara) and is inhibited in the presence of glucose and absence of arabinose. The mutant strains were grown for ~16 h under permissive or non-permissive conditions at 37°C.

- 26 -

Fig. S9. Westernblot analysis of TbPRORP2 expression in strains of (A) B. subtilis d7 and (B) E. coli BW. Expression of plasmid-encoded TbPRORP2 was analyzed in total protein extracts (T), as well as insoluble (IS) and soluble (S) protein fractions thereof, from B. subtilis d7[TbPRORP2] and E. coli BW[TbPRORP2]. Total protein from B. subtilis d7 and E. coli BW transformed with the empty vector pDG148(S/X)] was used as control. Protein fractions (30 µg per lane) were separated by 15% SDSPAGE and blotted onto PVDF membranes for detection with His-tag-specific monoclonal antibodies. Migration of marker proteins (M) and their sizes in kDa are indicated on the left. Putative full-length TbPRORP2 is indicated by the black arrow. (C) Corresponding analysis for AtPRORP1 expression in E. coli BW, demonstrating that AtPRORP1 is predominantly in the soluble protein fraction (panel C taken from Gobert et al., 2010).

- 27 -

REFERENCES Busch S, Kirsebom LA, Notbohm H, Hartmann RK (2000) Differential role of the intermolecular base-pairs G292-C(75) and G293-C(74) in the reaction catalyzed by Escherichia coli RNase P RNA. J Mol Biol., 299(4):941-951. Chang AC, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 134(3):11411156. Covarrubias L, Cervantes L, Covarrubias A, Soberón X, Vichido I, Blanco A, KupersztochPortnoy YM, Bolivar F (1981) Construction and characterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328. Gene 13(1):25-35. Damm, K., Bach S., Müller K.M., Klug G., Burenina O.Y., Kubareva E.A., Grünweller A., Hartmann R.K. (2015) Impact of RNA isolation protocols on RNA detection by Northern blotting. Methods Mol Biol., 1296, 29-38. Gobert A, Gutmann B, Taschner A, Gößringer M, Holzmann J, Hartmann RK, Rossmanith W, Giegé P (2010) A single Arabidopsis organellar protein has RNase P activity. Nat Struct Mol Biol. 17(6): 740-744. Gößringer M, Kretschmer-Kazemi Far R, Hartmann RK (2006) Analysis of RNase P protein (rnpA) expression in Bacillus subtilis utilizing strains with suppressible rnpA expression. J Bacteriol 188(19): 6816-6823. Gößringer M, Hartmann RK (2007) Function of heterologous and truncated RNase P proteins in Bacillus subtilis. Mol Microbiol 66(3): 801-813. Guyer, M.S., Reed, R.R., Steitz, J.A. and Low, K.B. 1981. Identification of a sexfactoraffinity site in E. coli as gamma delta. Cold Spring Harbor. Symp. Quant. Biol. 45: 135–140.

- 28 -

Hartmann,R.K., Toschka,H.Y., Erdmann,V.A. (1991) Processing and termination of 23S rRNA-5S rRNA-tRNA(Gly) primary transcripts in Thermus thermophilus HB8. J Bacteriol., 173, 2681-90. Howard MJ, Lim WH, Fierke CA, Koutmos M (2012) Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5' processing. Proc Natl Acad Sci USA 109(40): 16149-16154. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259), 680–685. Li D, Gößringer M, Hartmann RK (2011) Archaeal-bacterial chimeric RNase P RNAs: towards understanding RNA's architecture, function and evolution. Chembiochem 12(10): 1536-1543. Pavlova LV, Gößringer M, Weber C, Buzet A, Rossmanith W, Hartmann RK (2012) tRNA processing by protein-only versus RNA-based RNase P: kinetic analysis reveals mechanistic differences. Chembiochem 13(15): 2270-2276. Stragier P, Bonamy C, Karmazyn-Campelli C (1988) Processing of a sporulation sigma factor in Bacillus subtilis: how morphological structure could control gene expression. Cell 52(5): 697-704. Sun L, Campbell FE, Zahler NH, Harris ME (2006) Evidence that substrate-specific effects of C5 protein lead to uniformity in binding and catalysis by RNase P. EMBO J 25(17): 39984007. Taschner A, Weber C, Buzet A, Hartmann RK, Hartig A, Rossmanith W (2012) Nuclear RNase P of Trypanosoma brucei: a single protein in place of the multicomponent RNAprotein complex. Cell Rep 2(1): 19-25. Weber C, Hartig A, Hartmann RK, Rossmanith W (2014) Playing RNase P evolution: swapping the RNA catalyst for a protein reveals functional uniformity of highly divergent enzyme forms. PLoS Genet., 10(8), e1004506.

- 29 -

Wegscheid B, Hartmann RK (2006) The precursor tRNA 3'-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo. RNA 12(12): 2135-2148. Wegscheid B, Condon C, Hartmann RK (2006) Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences. EMBO Rep 7(4):411-417.