JOURNAL OF BACTERIOLOGY, Apr. 1991,
p.
Vol. 173, No. 8
2681-2690
0021-9193/91/082681-10$02.00/0 Copyright © 1991, American Society for Microbiology
Processing and Termination of 23S rRNA-5S rRNA-tRNAGlY Primary Transcripts in Thermus thermophilus HB8 ROLAND K. HARTMANN,* HOLGER Y. TOSCHKA, AND VOLKER A. ERDMANN Institut fur Biochemie, Freie Universitdt Berlin, 1000 Berlin 33, Federal Republic of Germany Received 26 October 1990/Accepted 12 February 1991
The two 23S rRNA-5S rRNA-tRNAGlY operons from the extreme thermophilic eubacterium Thermus thermophilus HB8 were used to characterize the in vivo processing and termination of 23S rRNA-5S rRNA-tRNAGlY primary transcripts in this organism by nuclease S1 mapping. A processing site in the pre-23S rRNA 3'-flanking region is located approximately 25 nucleotides upstream of 5S rRNA and precedes a putative 23S-5S rRNA spacer antitermination box A. Cleavage at this site and 5S rRNA 5' end formation were shown to be inseparable events. Termination of transcription at the uridine cluster following the terminationassociated hairpin was shown to be efficient but leaky. Subsequent to the operon, a functional promoter was detected whose -35 box coincided with the uridine-rich termination region. The promoter directed synthesis of a j8-galactosidase fusion protein in Escherichia coli.
In the extreme thermophilic eubacterium Thermus thermophilus, each rRNA gene is present in two copies (13). T. thermophilus is the first prokaryotic organism whose 16S rRNA genes have been shown to be locally and transcriptionally separated from the 23S-5S rRNA genes (12, 13, 15). Recent reports have given evidence of similar rRNA gene organization in the planctomycete Pirellula marina (23). Processing of rRNA primary transcripts in bacteria is best understood in Escherichia coli (1, 20, 29); significantly less is known for other prokaryotes. As an extension to our previous work, we have characterized the in vivo maturation of primary transcripts in the 23S rRNA 3'-flanking, 5S rRNA, and tRNAGlY coding regions, as well as the sequences involved in transcriptional termination. Such studies are a prerequisite for identification and characterization of activities involved in processing and termination of rRNA primary transcripts in the extreme thermophilic eubacterium.
laqIq lacZ AM15] was used as the host strain for M13 and the plasmid pKK232-8, as well as for expression of the ,Bgalactosidase fusion protein. E. coli MC 1061 (26, 32) araD139 A(araABC-leu)7679, AlacX74 galU galK rpsL thi hsdR mcrB was also used for expression of the P-galactosidase fusion protein. Bulk tRNA (Boehringer Mannheim) from the ribonuclease I-deficient E. coli strain MRE 600 (ATCC 29417) (40) was used as the control in nuclease Si protection analyses. Cloning of the two 23S rRNA-5S rRNA-tRNAGlY operons. Cloning was performed as described previously (13). The recombinant plasmids pTT675 and pTT700 each represent one of two 23S rRNA-5S rRNA-tRNAGlY operons. Both operons are identical in the 23S rRNA-5S rRNA-tRNAGlY coding regions, but they begin to diverge at the putative hairpin terminator region (data not shown). Plasmids. In addition to the plasmids pTT675 and pTT700, the pBR322 derivative pKK232-8 (3) was used for identification of the promoter following the terminator of the 23S rRNA-5S rRNA-tRNAGly operon. The vector carries a constitutively expressed ,-lactamase gene and a promoterdeficient chloramphenicol acetyltransferase (CAT) gene preceded by an M13 mp8 multicloning site. Insertion of promoter-carrying DNA fragments into the cloning site leads to expression of the chloramphenicol resistance gene in E. coli cells. The E. coli plasmid pNM481 (26), utilized for expression of lacZ fusion proteins, harbors a constitutively expressed 1-lactamase gene and a promoter-deficient lacZ gene followed by the lacY gene. An M13 mp8 multicloning site was fused to the eighth codon of the lacZ gene. In order to express P-galactosidase fusion proteins, fragments carrying the promoter and the 5' portion of a protein gene must be inserted into the multicloning site in frame with the lacZ gene. For adapting the two other reading frames, otherwise identical vectors (pNM480 and pNM482) were constructed (26). 5' and 3' labeling of DNA fragments used for nuclease S1 protection analysis. The 500-bp AvaI-BamHI fragment derived from the recombinant plasmid pTT700 was 3' end labeled exclusively at the AvaI site by filling in the recessed 3' ends with [x-32P]dCTP in the absence of dATP and dTTP by utilizing Klenow polymerase (24). This fragment covered
MATERIALS AND METHODS
Enzymes were purchased from Boehringer Mannheim, Pharmacia, New England Biolabs, and Bethesda Research Laboratories. Radioactive nucleotides were obtained from Amersham. Primer synthesis and DNA sequencing according to the M13-dideoxy method (33) were performed as described (12, 15). Bacterial strains. T. thermophilus HB8, an extremely thermophilic, gram-negative, nonmotile, nonsporulating, rod-shaped and obligatory aerobic eubacterium containing the cryptic plasmids pTT8 and pVV8 (38) was isolated from a Japanese hot spring (28) and kindly provided by T. Oshima. Microbiological analyses of this strain and numerous other gram-negative hot-water isolates have been reported (6, 19). Cells (ATCC 27634) were grown at 70 to 75°C in medium D, as previously described (4), supplemented with 5 g of tryptone (Difco Laboratories) per liter, 4 g of yeast extract per liter, 2 g of NaCl per liter, and 1 g of glucose per liter. Cells were harvested in the mid-log phase. E. coli BMH 71/18 (21) supE thi A(lac-proAB) F'[proAB+ *
Corresponding author. 2681
2682
approximately 85 nucleotides of 3' 23S rDNA, the remainder of the operon, and 98 nucleotides of sequence following the putative hairpin terminator (Fig. 1B, probe 1). All other probes were derived from the pTT675-encoded operon. The 218-bp PvuII-StyI and the 129-bp PvuII-RsaI fragments (Fig. 1B, probes 2 and 3) were dephosphorylated and labeled at their 5' ends with T4 polynucleotide kinase
(25).
JJ. BACTERIOL.
HARTMANN ET AL.
The 464-bp PvuII-Asp 718 and the 352-bp PvuII-XbaI fragments (Fig. 1B, probes 8 and 9) were dephosphorylated and 5' labeled as larger PstI-XbaI and PstI-Asp 718 fragments, followed by PvuII cleavage and preparative gel electrophoresis to ascertain that both probes were carrying only one 5' 32P label at the XbaI or Asp 718 site, respectively. The above-mentioned PstI site was located in the 23S rRNA coding region, 840 bp upstream of the PvuII site shown in Fig. 1B. The 223-bp RsaI-XbaI, 180-bp RsaI-HpaII, 130-bp StyIXbaI, and 129-bp PvuII-RsaI fragments (Fig. 1B, probes 4 to 7) all were labeled with [a-32P]dCTP by utilizing T4 DNA polymerase as described previously (12). The method yields thoroughly labeled probes whose 32P incorporation is highest at the 3' ends and decreases toward the 5' ends. Nuclease Si mapping. Preparation of total RNA from exponentially growing T. thermophilus cells and nuclease S1 protection analyses were performed as described (16). The hybridization temperatures were 37, 50, and 60°C. In controls, equal amounts (100 ,ug) of bulk tRNA from E. coli MRE 600 instead of total RNA from T. thermophilus were incubated with the labeled DNA probe. Expression of the 13-galactosidase fusion protein. The 337-bp RsaI-RsaI fragment, ranging from the RsaI site in the 5S rRNA gene to the operon distal RsaI site (which is part of an Asp 718 site) (Fig. 1B), was cloned into the SmaI site of the lacZ gene fusion vector pNM481 (26). Recombinant plasmids were introduced into E. coli BMH 71/18 (21) carrying a truncated ,3-galactosidase gene (lacZ AM15) and into the lac-deficient E. coli MC 1061 (26), kindly provided by Walter Messer. Positive clones were identified by their light blue color, and the orientation of the insert was checked by cutting at the asymmetric XbaI site (Fig. 1B) of the insert and at the EcoRI site of the pNM481 multicloning site. Cell extracts were run on sodium dodecyl sulfate (SDS)-8% polyacrylamide gels (36), and the gels were transferred to
derivatized nitrocellulose (Nitro Screen West, Dupont) by semidry electrophoretic transfer (22) utilizing a Pharmacia device (2117 Multiphor II). After blotting, the filter was incubated overnight at room temperature in TBS-T buffer (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% [vol/vol] Tween 20) supplemented with 5% (wt/vol) nonfat powdered milk (Amersham). The filter was washed three times with TBS-T buffer and twice with TBS buffer (TBS-T buffer without Tween 20), followed by incubation for 60 min at room temperature in a solution containing 500 ng of monoclonal mouse anti-p-galactosidase antibodies per ml (Boehringer), 6% nonfat powdered milk, and 50 ,uM sodium azide. The filter was washed three times with TBS-T buffer and twice with TBS buffer. Immunostaining (11) was accomplished with an anti-mouse immunoglobulin G-peroxidase conjugate (Amersham). RESULTS
The two large ribosomal subunit RNA operons from T. thermophilus harbor a 23S rRNA, a SS rRNA, and a tRNAGlY gene, followed by a potential hairpin structure (Fig. 1A). The structural organization of T. thermophilus 23S rRNA-5S rRNA-tRNAGly operons is similar to that found in the 23S rRNA distal regions of E. coli rrnC, rrnD, and rrnH operons (7, 35, 42). One of the two T. thermophilus operons is followed by a potential protein gene (Fig. 4). Similarly, a putative promoter followed by an open reading frame was reported for E. coli rrnH 50 bp downstream from the terminator hairpin (35). Pre-23S rRNA 5'-flanking and 23S rRNA-5S rRNA intergenic spacer sequences of primary transcripts from T. thermophilus (Fig. 1A) are of reduced size and therefore lack the extensive base pairing found in E. coli (2) or Pseudomonas aeruginosa pre-23S rRNAs (37a). For T. thermophilus pre23S rRNA flanking sequences, a potential arrangement of alternating hairpins, internal loops, and shorter helical segments was observed (Fig. 1A). Nuclease Si mapping. (i) Pre-23S rRNA 3'-flanking region. To detect 3' precursors in the 3'-flanking region of pre-23S rRNA, a 500-bp AvaI-BamHI fragment comprising approximately 85 nucleotides of 3' 23S rDNA was 3' end labeled exclusively at the AvaI site (Fig. 1B, probe 1), denatured,
FIG. 1. (A) Potential secondary structure of transcripts from the two 23S rRNA-5S rRNA-tRNAGly operons (pTT675 and pTT700) of T. thermophilus HB8. Processing sites (indicated by arrows) surrounding the 23S rRNA have been reported earlier (16). Processing sites downstream from the indicated PvuII site were deduced from data shown in Fig. 2, 3, and 5. Numbered circles on each arrow indicate the corresponding probe (specified in panel B) used to detect the cleavage sites; thick arrows indicate nuclease Si protection signals of relatively high intensity. An open box marks a direct repeat, and a hatched box indicates a leader and spacer box A sequence probably involved in antitermination (12). The continuous numbering system of the primary transcript begins with the start of transcription (15), indicated by an open triangle (position +1), and considers the 23S rRNA (18) and 5S rRNA (39) coding sequences. 5' and 3' ends of 5S rRNA, deduced from RNA sequencing data (41), are indicated; additional 5S rRNA species shortened by one or two nucleotides at their 5' ends and/or one nucleotide at their 3' ends were observed (41). The framed inlay shows an alternative secondary structural folding in the region confined by nucleotides 3 to 27 and 3018 to 3042 of the primary transcript. In this secondary structural context, cleavage sites at nucleotides 3038 and 3039 display some resemblance to RNase III cleavage sites in E. coli pre-rRNA (2). (B) Schematic representation of the region downstream from the 23S rRNA and DNA probes used for Si nuclease mapping. The sequences of both operons (data not shown) differ downstream from the terminator hairpins (T). At the top, the solid line indicates the pTT675-encoded operon and the broken line indicates the pTT700-encoded operon. The second RsaI site, not utilized for nuclease Si protection analyses and therefore indicated in parentheses, is part of the Asp 718 site; cleavage at this RsaI site and at the upstream RsaI site, located in the 5S rRNA coding sequence, yielded a 337-bp Rsal-RsaI blunt-ended fragment that was cloned into the SmaI site of the lacZ fusion expression vector pNM481 (see Materials and Methods and Fig. 4). Asterisks indicate "P-labeled nucleotides. Probe 1, the only probe derived from the pTT700 operon, was "P-labeled by filling in the recessed 3' end of the Aval site with Klenow polymerase. Probes 2, 3, 8, and 9 were prepared by the 5' end labeling of the StyI, RsaI, Asp 718, and XbaI sites, respectively. Probes 4 (RsaI-XbaI), 5 (RsaI-HpaII), 6 (StyI-XbaI), and 7 (PvuII-RsaI) were labeled by T4 DNA polymerase as described (12), yielding thoroughly labeled probes whose 32p incorporation decreases toward the 5' ends, as indicated by the discontinuous distribution of asterisks.
T. THERMOPHILUS 23S rRNA-55 rRNA-tRNAG0y TRANSCRIPTS
VOL. 173, 1991
27
3018
A
GeC
G-C GG C CG-C ALU U A G-C GC
G-C
G^C
G.,dG USA
G
T
CG
3-G
G03042
pTT 675 UGCUUUG
a A
GeoC AOU
-.
coGo' U 3406U
coGo'
COG
coG AAACOGUUUAUCCUUUUA
A A
tRNA'* Ava
Pl
Rsal
Xbal
Stl
Asp 718
~~~~~~~~~~(pTT700)(R sa I)
_\
0 0@ 3'*
3' *-* *
*
*
--*
*_
2683
2684
HARTMANN ET AL.
MMMM M MM 1 2 34 5 1 2 3 4 5 6 7
*FC ,At|.'.::'so
J. BACTERIOL.
C Ml 2 34 5 M n
._
1 2 3 M
*_
391o 327 3 33x.-' 3318e S315 297b" 295a
to
.s
_ :
..
JC
-~266a._
:
*=8,
174s b 1666is r
H
H
1:.
p.
Vol. 173, No. 8
2681-2690
0021-9193/91/082681-10$02.00/0 Copyright © 1991, American Society for Microbiology
Processing and Termination of 23S rRNA-5S rRNA-tRNAGlY Primary Transcripts in Thermus thermophilus HB8 ROLAND K. HARTMANN,* HOLGER Y. TOSCHKA, AND VOLKER A. ERDMANN Institut fur Biochemie, Freie Universitdt Berlin, 1000 Berlin 33, Federal Republic of Germany Received 26 October 1990/Accepted 12 February 1991
The two 23S rRNA-5S rRNA-tRNAGlY operons from the extreme thermophilic eubacterium Thermus thermophilus HB8 were used to characterize the in vivo processing and termination of 23S rRNA-5S rRNA-tRNAGlY primary transcripts in this organism by nuclease S1 mapping. A processing site in the pre-23S rRNA 3'-flanking region is located approximately 25 nucleotides upstream of 5S rRNA and precedes a putative 23S-5S rRNA spacer antitermination box A. Cleavage at this site and 5S rRNA 5' end formation were shown to be inseparable events. Termination of transcription at the uridine cluster following the terminationassociated hairpin was shown to be efficient but leaky. Subsequent to the operon, a functional promoter was detected whose -35 box coincided with the uridine-rich termination region. The promoter directed synthesis of a j8-galactosidase fusion protein in Escherichia coli.
In the extreme thermophilic eubacterium Thermus thermophilus, each rRNA gene is present in two copies (13). T. thermophilus is the first prokaryotic organism whose 16S rRNA genes have been shown to be locally and transcriptionally separated from the 23S-5S rRNA genes (12, 13, 15). Recent reports have given evidence of similar rRNA gene organization in the planctomycete Pirellula marina (23). Processing of rRNA primary transcripts in bacteria is best understood in Escherichia coli (1, 20, 29); significantly less is known for other prokaryotes. As an extension to our previous work, we have characterized the in vivo maturation of primary transcripts in the 23S rRNA 3'-flanking, 5S rRNA, and tRNAGlY coding regions, as well as the sequences involved in transcriptional termination. Such studies are a prerequisite for identification and characterization of activities involved in processing and termination of rRNA primary transcripts in the extreme thermophilic eubacterium.
laqIq lacZ AM15] was used as the host strain for M13 and the plasmid pKK232-8, as well as for expression of the ,Bgalactosidase fusion protein. E. coli MC 1061 (26, 32) araD139 A(araABC-leu)7679, AlacX74 galU galK rpsL thi hsdR mcrB was also used for expression of the P-galactosidase fusion protein. Bulk tRNA (Boehringer Mannheim) from the ribonuclease I-deficient E. coli strain MRE 600 (ATCC 29417) (40) was used as the control in nuclease Si protection analyses. Cloning of the two 23S rRNA-5S rRNA-tRNAGlY operons. Cloning was performed as described previously (13). The recombinant plasmids pTT675 and pTT700 each represent one of two 23S rRNA-5S rRNA-tRNAGlY operons. Both operons are identical in the 23S rRNA-5S rRNA-tRNAGlY coding regions, but they begin to diverge at the putative hairpin terminator region (data not shown). Plasmids. In addition to the plasmids pTT675 and pTT700, the pBR322 derivative pKK232-8 (3) was used for identification of the promoter following the terminator of the 23S rRNA-5S rRNA-tRNAGly operon. The vector carries a constitutively expressed ,-lactamase gene and a promoterdeficient chloramphenicol acetyltransferase (CAT) gene preceded by an M13 mp8 multicloning site. Insertion of promoter-carrying DNA fragments into the cloning site leads to expression of the chloramphenicol resistance gene in E. coli cells. The E. coli plasmid pNM481 (26), utilized for expression of lacZ fusion proteins, harbors a constitutively expressed 1-lactamase gene and a promoter-deficient lacZ gene followed by the lacY gene. An M13 mp8 multicloning site was fused to the eighth codon of the lacZ gene. In order to express P-galactosidase fusion proteins, fragments carrying the promoter and the 5' portion of a protein gene must be inserted into the multicloning site in frame with the lacZ gene. For adapting the two other reading frames, otherwise identical vectors (pNM480 and pNM482) were constructed (26). 5' and 3' labeling of DNA fragments used for nuclease S1 protection analysis. The 500-bp AvaI-BamHI fragment derived from the recombinant plasmid pTT700 was 3' end labeled exclusively at the AvaI site by filling in the recessed 3' ends with [x-32P]dCTP in the absence of dATP and dTTP by utilizing Klenow polymerase (24). This fragment covered
MATERIALS AND METHODS
Enzymes were purchased from Boehringer Mannheim, Pharmacia, New England Biolabs, and Bethesda Research Laboratories. Radioactive nucleotides were obtained from Amersham. Primer synthesis and DNA sequencing according to the M13-dideoxy method (33) were performed as described (12, 15). Bacterial strains. T. thermophilus HB8, an extremely thermophilic, gram-negative, nonmotile, nonsporulating, rod-shaped and obligatory aerobic eubacterium containing the cryptic plasmids pTT8 and pVV8 (38) was isolated from a Japanese hot spring (28) and kindly provided by T. Oshima. Microbiological analyses of this strain and numerous other gram-negative hot-water isolates have been reported (6, 19). Cells (ATCC 27634) were grown at 70 to 75°C in medium D, as previously described (4), supplemented with 5 g of tryptone (Difco Laboratories) per liter, 4 g of yeast extract per liter, 2 g of NaCl per liter, and 1 g of glucose per liter. Cells were harvested in the mid-log phase. E. coli BMH 71/18 (21) supE thi A(lac-proAB) F'[proAB+ *
Corresponding author. 2681
2682
approximately 85 nucleotides of 3' 23S rDNA, the remainder of the operon, and 98 nucleotides of sequence following the putative hairpin terminator (Fig. 1B, probe 1). All other probes were derived from the pTT675-encoded operon. The 218-bp PvuII-StyI and the 129-bp PvuII-RsaI fragments (Fig. 1B, probes 2 and 3) were dephosphorylated and labeled at their 5' ends with T4 polynucleotide kinase
(25).
JJ. BACTERIOL.
HARTMANN ET AL.
The 464-bp PvuII-Asp 718 and the 352-bp PvuII-XbaI fragments (Fig. 1B, probes 8 and 9) were dephosphorylated and 5' labeled as larger PstI-XbaI and PstI-Asp 718 fragments, followed by PvuII cleavage and preparative gel electrophoresis to ascertain that both probes were carrying only one 5' 32P label at the XbaI or Asp 718 site, respectively. The above-mentioned PstI site was located in the 23S rRNA coding region, 840 bp upstream of the PvuII site shown in Fig. 1B. The 223-bp RsaI-XbaI, 180-bp RsaI-HpaII, 130-bp StyIXbaI, and 129-bp PvuII-RsaI fragments (Fig. 1B, probes 4 to 7) all were labeled with [a-32P]dCTP by utilizing T4 DNA polymerase as described previously (12). The method yields thoroughly labeled probes whose 32P incorporation is highest at the 3' ends and decreases toward the 5' ends. Nuclease Si mapping. Preparation of total RNA from exponentially growing T. thermophilus cells and nuclease S1 protection analyses were performed as described (16). The hybridization temperatures were 37, 50, and 60°C. In controls, equal amounts (100 ,ug) of bulk tRNA from E. coli MRE 600 instead of total RNA from T. thermophilus were incubated with the labeled DNA probe. Expression of the 13-galactosidase fusion protein. The 337-bp RsaI-RsaI fragment, ranging from the RsaI site in the 5S rRNA gene to the operon distal RsaI site (which is part of an Asp 718 site) (Fig. 1B), was cloned into the SmaI site of the lacZ gene fusion vector pNM481 (26). Recombinant plasmids were introduced into E. coli BMH 71/18 (21) carrying a truncated ,3-galactosidase gene (lacZ AM15) and into the lac-deficient E. coli MC 1061 (26), kindly provided by Walter Messer. Positive clones were identified by their light blue color, and the orientation of the insert was checked by cutting at the asymmetric XbaI site (Fig. 1B) of the insert and at the EcoRI site of the pNM481 multicloning site. Cell extracts were run on sodium dodecyl sulfate (SDS)-8% polyacrylamide gels (36), and the gels were transferred to
derivatized nitrocellulose (Nitro Screen West, Dupont) by semidry electrophoretic transfer (22) utilizing a Pharmacia device (2117 Multiphor II). After blotting, the filter was incubated overnight at room temperature in TBS-T buffer (20 mM Tris-HCl [pH 7.6], 137 mM NaCl, 0.1% [vol/vol] Tween 20) supplemented with 5% (wt/vol) nonfat powdered milk (Amersham). The filter was washed three times with TBS-T buffer and twice with TBS buffer (TBS-T buffer without Tween 20), followed by incubation for 60 min at room temperature in a solution containing 500 ng of monoclonal mouse anti-p-galactosidase antibodies per ml (Boehringer), 6% nonfat powdered milk, and 50 ,uM sodium azide. The filter was washed three times with TBS-T buffer and twice with TBS buffer. Immunostaining (11) was accomplished with an anti-mouse immunoglobulin G-peroxidase conjugate (Amersham). RESULTS
The two large ribosomal subunit RNA operons from T. thermophilus harbor a 23S rRNA, a SS rRNA, and a tRNAGlY gene, followed by a potential hairpin structure (Fig. 1A). The structural organization of T. thermophilus 23S rRNA-5S rRNA-tRNAGly operons is similar to that found in the 23S rRNA distal regions of E. coli rrnC, rrnD, and rrnH operons (7, 35, 42). One of the two T. thermophilus operons is followed by a potential protein gene (Fig. 4). Similarly, a putative promoter followed by an open reading frame was reported for E. coli rrnH 50 bp downstream from the terminator hairpin (35). Pre-23S rRNA 5'-flanking and 23S rRNA-5S rRNA intergenic spacer sequences of primary transcripts from T. thermophilus (Fig. 1A) are of reduced size and therefore lack the extensive base pairing found in E. coli (2) or Pseudomonas aeruginosa pre-23S rRNAs (37a). For T. thermophilus pre23S rRNA flanking sequences, a potential arrangement of alternating hairpins, internal loops, and shorter helical segments was observed (Fig. 1A). Nuclease Si mapping. (i) Pre-23S rRNA 3'-flanking region. To detect 3' precursors in the 3'-flanking region of pre-23S rRNA, a 500-bp AvaI-BamHI fragment comprising approximately 85 nucleotides of 3' 23S rDNA was 3' end labeled exclusively at the AvaI site (Fig. 1B, probe 1), denatured,
FIG. 1. (A) Potential secondary structure of transcripts from the two 23S rRNA-5S rRNA-tRNAGly operons (pTT675 and pTT700) of T. thermophilus HB8. Processing sites (indicated by arrows) surrounding the 23S rRNA have been reported earlier (16). Processing sites downstream from the indicated PvuII site were deduced from data shown in Fig. 2, 3, and 5. Numbered circles on each arrow indicate the corresponding probe (specified in panel B) used to detect the cleavage sites; thick arrows indicate nuclease Si protection signals of relatively high intensity. An open box marks a direct repeat, and a hatched box indicates a leader and spacer box A sequence probably involved in antitermination (12). The continuous numbering system of the primary transcript begins with the start of transcription (15), indicated by an open triangle (position +1), and considers the 23S rRNA (18) and 5S rRNA (39) coding sequences. 5' and 3' ends of 5S rRNA, deduced from RNA sequencing data (41), are indicated; additional 5S rRNA species shortened by one or two nucleotides at their 5' ends and/or one nucleotide at their 3' ends were observed (41). The framed inlay shows an alternative secondary structural folding in the region confined by nucleotides 3 to 27 and 3018 to 3042 of the primary transcript. In this secondary structural context, cleavage sites at nucleotides 3038 and 3039 display some resemblance to RNase III cleavage sites in E. coli pre-rRNA (2). (B) Schematic representation of the region downstream from the 23S rRNA and DNA probes used for Si nuclease mapping. The sequences of both operons (data not shown) differ downstream from the terminator hairpins (T). At the top, the solid line indicates the pTT675-encoded operon and the broken line indicates the pTT700-encoded operon. The second RsaI site, not utilized for nuclease Si protection analyses and therefore indicated in parentheses, is part of the Asp 718 site; cleavage at this RsaI site and at the upstream RsaI site, located in the 5S rRNA coding sequence, yielded a 337-bp Rsal-RsaI blunt-ended fragment that was cloned into the SmaI site of the lacZ fusion expression vector pNM481 (see Materials and Methods and Fig. 4). Asterisks indicate "P-labeled nucleotides. Probe 1, the only probe derived from the pTT700 operon, was "P-labeled by filling in the recessed 3' end of the Aval site with Klenow polymerase. Probes 2, 3, 8, and 9 were prepared by the 5' end labeling of the StyI, RsaI, Asp 718, and XbaI sites, respectively. Probes 4 (RsaI-XbaI), 5 (RsaI-HpaII), 6 (StyI-XbaI), and 7 (PvuII-RsaI) were labeled by T4 DNA polymerase as described (12), yielding thoroughly labeled probes whose 32p incorporation decreases toward the 5' ends, as indicated by the discontinuous distribution of asterisks.
T. THERMOPHILUS 23S rRNA-55 rRNA-tRNAG0y TRANSCRIPTS
VOL. 173, 1991
27
3018
A
GeC
G-C GG C CG-C ALU U A G-C GC
G-C
G^C
G.,dG USA
G
T
CG
3-G
G03042
pTT 675 UGCUUUG
a A
GeoC AOU
-.
coGo' U 3406U
coGo'
COG
coG AAACOGUUUAUCCUUUUA
A A
tRNA'* Ava
Pl
Rsal
Xbal
Stl
Asp 718
~~~~~~~~~~(pTT700)(R sa I)
_\
0 0@ 3'*
3' *-* *
*
*
--*
*_
2683
2684
HARTMANN ET AL.
MMMM M MM 1 2 34 5 1 2 3 4 5 6 7
*FC ,At|.'.::'so
J. BACTERIOL.
C Ml 2 34 5 M n
._
1 2 3 M
*_
391o 327 3 33x.-' 3318e S315 297b" 295a
to
.s
_ :
..
JC
-~266a._
:
*=8,
174s b 1666is r
H
H
1:.
