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Why is processing of 23 S ribosomal RNA in Escherichia coli not obligate for its function?
J. MoZ. Biol. (1985) 186, 669-672
Why is Processing of 23 S Ribosomal RNA in Escherichia coli not Obligate for its Function? In an RNase III-deficient mutant of Escherichia coli, all 23 S ribosomal RNA in ribosomes is present in an unprocessed form with a double-stranded stem at the base of the molecule stable enough to be detected by electron microscopy under conditions where all other secondary structure is denatured. Molecules with variable stem lengths enter freely into polysomes, consistent with the existence of a similar but much shorter stem in mature 23 S rRNA in wild-type ribosomes.
The primary transcript of Escherichia coli ribosomal RNA (“30 S pre-rRNA”) includes 16 S, 23 S and 5 S rRNA sequences (Dunn & Studier, 1973; Nikolaev et al., 1973). Ordinarily these are rapidly cleaved apart during transcription (King & Schlessinger, 1984). The cleavages occur in strongly base-paired stems which enclose large loops of the 16 S and 23 S rRNA sequences (Young & Steitz, 1978; Bram et al., 1980). These sites are recognized by RNase III, which is specific for double-stranded RNA (Robertson et al., 1968). In an RNase IIIdeficient mutant ABLl (Kindler et al., 1973) the cleavages fail. Mature 16 S rRNA is nevertheless formed in the mutant at wild-type rates, apparently by the direct formation of mature termini without preliminary cleavage in the double-stranded stems (King & Schlessinger, 1984). In contrast, all the molecules of 23 S rRNA accumulate in ribosomes in an unprocessed form, with up to 100 bases of precursor sequence at both their 5’ and 3’ ends (King et al., 1984). The continued presence of a double-stranded stem in the 50 S ribosomes of the mutant strain was strongly implied by the observation that RNase III cleaved isolated 23 S pre-rRNA and that in the ribosomes at the same points (Sirdeshmukh et al., 1985). The structure can be observed by electron microscopy in conditions denaturing enough to eliminate all but the most stable features of secondary structure. In the conditions used for Figure 1, for example, mature 23 S rRNA molecules all appear as a linear structure with none or a few internal loops (Klein et al., 1983). Instead, the rRNA molecules from strain ABLl look more complex. In 30 to 40% of all completely traceable molecules in three independent experiments, the short stem is the only feature of secondary structure remaining (as in the two molecules in Fig. l(a)), with the RNA chain held together in a large loop. In some other molecules, internal loops make the total contour difficult to trace; but at least 70% of the full-length molecules are circular, with a short stem visible in most of them (Fig. l(b)). Several precursor species were seen in the total RXA of the RNase III-deficient strain; and some, 0022%2836/85/230669-04
$03.00/O
but perhaps not all, of these speciesmust be active. To test this, the 5’ and 3’ termini of 23 S rRNA in 50 S ribosomes from the mutant were compared to those in polysomes. Figure 2 shows that the same termini were found in all the fractions examined. The 5’ end of 23 S rRNA in polysomes and 70 S and 50 S ribosomes from ABLl included species A, Cl and C2 (as described by King et al., 1984) up to 97 nucleotides longer than the mature terminus (lanes 2 to 4). Lane 1 shows the termini in the total RNA of the wild-type strain DlO (Gasteland, 1966). At the 3’ end (Fig. 2, lanes 6 to 8), two major species, A and C (see King et al., 1984), are up to 95 nucleotides longer than the published wild-type terminus (compare with lane 5) and are seen at comparable levels in 50 S ribosomes and in polysomes of the mutant. The stem at the base of the mutant rRNA has a AG” value of the order of - 100 kcal (1 kcal = 4.184 kJ), and a resulting stabilization much greater than other interactions in 23 S rRNA (Klein et al., 1983). Base-paired stems of intermediate stability can be formed in various regions of heated rRNA, and are stable even at 55°C in 70% formamide (Klein et aZ., 1985); the stem at the base of 23 S rRNA is thus even more stable. It therefore seemsvery likely that the 50 S ribosomes of the mutant strain function throughout the cycle of protein synthesis with the 5’ and 3’ termini of 23 S rRNA base-paired. This suggestion is strongest for the longest RNA chains; but because molecules with shorter stems are not disfavored in the population in polysomes, species with all possible lengths must be comparably functional. A short double-stranded stem (8 nucleotides long) has been inferred in models for the secondary structure of the mature 23 S rRNA (Noller et al., 1981), though its existence in ribosomes has not been verified and there is also no information about whether the putative stem might remain intact during protein synthesis. Our results make it increasingly likely that such a structure exists in wild-type ribosomes, though it is not stable enough to hold together in the conditions used for electron microscopy (Klein et al., 1983). The functionality of the 50 S ribosomes with long base-paired tails of
669
0
1985
Academic
Press
Inc.
(London)
Ltd.
670
R. Sirdeshmukh
and D. Schlessinger
Figure 1. Electron micrograph of a 23 S rRNA molecule from a 50 S ribosome of strain ABLl. 50 S ribosomes were dissolved in a 0.2% (w/v) sodium dodecyl sulfate, 10 mlcr-Tris. HCl (pH 7.4), 5 m&x-Mgcl,, and electrophoresed in a l”/b (w/v) agarose gel with Tris-acetate (pH 79). 5 mM-MgC1, as running buffer. The rRPu‘A was eluted from the gel slice in a buffer (Saha et aZ., 1983) containing 5 mM-Mgcl,, precipitated with cold ethanol, and dissolved and spread in 10 rnzxTris’ HCl (pH 7.4), 10 mM-EDTA, and 50% formamide (Klein et al., 1983). The 2 molecules labeled (a) are magnified 96,700 x The field at the right, labeled (b) is magnified 56,lOOO x ; the contour length of the molecule in (b) is the same (+O.l pm) as that in (a).
671
Letters to the Editor
1 Figure 2. Si nuclease analysis of the 5’ (left) and 3’ termini (right) of 23 S rRTu’A from strains DlO and ABLl’. Total R?jA from control strain DlO or RPU’A from polysomes or 70 S or 50 S ribosomes of strain ABLl was hybridized with single-stranded end-labeled DXA probes for the respective termini and S1 protection analysis assessed as described by King et al. (1984). The positions of the mature terminus M and the unprocessed species A, C, Cl and C2 are indicated (see King et al., 1984). Lanes 1 and 5, strain DlO RNA; lanes 2 and 6! ABLl polysomal RNA; lanes 3 and 7. RKA from ABLl 70 S ribosomes; lanes 4 and 8, RXA from ABLl 50 S ribosomes.
variable length in strain ABLl can therefore be rationalized if the termini of mature 23 S rRNA molecules are also base-paired and remain in close proximity in ribosomes during translation. The 3’ terminus of 23 S rRNA has been located at a point opposite the Ll binding site on the 50 S ribosome (Shatsky et al., 1980; Stoffler-Meilicke et al., 1981), and our results suggest that the 5’ terminus is located at the same point. Apparently any extra RNA sequences at this location block neither peptide
bond
formation
nor translocation.
We thank Grady Phillips and Tom Rucinsky for their extensive help in electron microscopy. These studies were supported in part by Kational Science Foundation grant PCM-8017402.
Ravi Sirdeshmukh David Schlessinger Department of Microbiology and Immunology Washington University School of Medicine St Louis, MO 63110, U.S.A. Received 14 May 1985, and in revised form 9 August 1985
References Bram, R. J., Young, R. A. & Steitz, J. A. (1980). Cell, 19, 393-401. Dunn, J. J. & Studier, F. W. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3296-3300. Gasteland, R. F. (1966). J. Mol. Biol. 16, 67-84. Kindler, P., Keil, T. & Hofschneider, P. (1973). Mol. Gen. Genet. 126, 53-69. King, T. C. & Schlessinger, D. (1984). J. Biol. Chem. 258, 12034-12042. King, T. C., Sirdeshmukh, R. & Schlessinger, D. (1984). Proc. Nat. Acad. Sci., U.S.A. 81, 185-188. Klein, B. K., King, T. C. & Schlessinger, D. (1983). J. Mol. Biol. 168, 809-830. Klein, B. K., Staden, A. & Schlessinger, D. (1985). Proc. Nat. Acud. Sci., U.S.A. 82, 3539-3542. Wikolaev, N., Silengo, L. & Schlessinger, D. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3361-3365. Pu’oller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, R. R., Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A., Gupta, R. & Woese, C. R. (1981). Nucl. Acids Rex 9, 6167-6189. Robertson, H. D., Webster, R. E. & Zinder. N. D. (1968). J. Biol. Chem. 243, 82-91. Saha, B., Strelow, S. t Schlessinger, D. (1983). J. B&hem. Biophys. Meth. 7, 277-284.
672
R. Sirdeshmukh and D. Schlessinger
Shatsky, I. pu’., Evstafieva, A. G., Bystrova, T. F.. Bogdanov, A. A. 6 Vasiliev, V. D. (1980). FEBS Letters, 122, 251-255. Sirdeshmukh, R. & Schlessinger, D. (1985). NucE. Acids Res. 13, 5041-5054.
Stoffler-Meilicke, M., Stoffler, G.. Odom, 0. W.. Zinn, 9.. Kramer, G. & Hardesty, B. (1981). Proc. Nat. Acad. Sci., U.S.A. 78, 5538-5542. Young, R. & St&z, J. (1978). Proc. LVat. Acad. Sci., U.S.A. 75, 3593-3597.
Edited by S. Brenner
Why is Processing of 23 S Ribosomal RNA in Escherichia coli not Obligate for its Function? In an RNase III-deficient mutant of Escherichia coli, all 23 S ribosomal RNA in ribosomes is present in an unprocessed form with a double-stranded stem at the base of the molecule stable enough to be detected by electron microscopy under conditions where all other secondary structure is denatured. Molecules with variable stem lengths enter freely into polysomes, consistent with the existence of a similar but much shorter stem in mature 23 S rRNA in wild-type ribosomes.
The primary transcript of Escherichia coli ribosomal RNA (“30 S pre-rRNA”) includes 16 S, 23 S and 5 S rRNA sequences (Dunn & Studier, 1973; Nikolaev et al., 1973). Ordinarily these are rapidly cleaved apart during transcription (King & Schlessinger, 1984). The cleavages occur in strongly base-paired stems which enclose large loops of the 16 S and 23 S rRNA sequences (Young & Steitz, 1978; Bram et al., 1980). These sites are recognized by RNase III, which is specific for double-stranded RNA (Robertson et al., 1968). In an RNase IIIdeficient mutant ABLl (Kindler et al., 1973) the cleavages fail. Mature 16 S rRNA is nevertheless formed in the mutant at wild-type rates, apparently by the direct formation of mature termini without preliminary cleavage in the double-stranded stems (King & Schlessinger, 1984). In contrast, all the molecules of 23 S rRNA accumulate in ribosomes in an unprocessed form, with up to 100 bases of precursor sequence at both their 5’ and 3’ ends (King et al., 1984). The continued presence of a double-stranded stem in the 50 S ribosomes of the mutant strain was strongly implied by the observation that RNase III cleaved isolated 23 S pre-rRNA and that in the ribosomes at the same points (Sirdeshmukh et al., 1985). The structure can be observed by electron microscopy in conditions denaturing enough to eliminate all but the most stable features of secondary structure. In the conditions used for Figure 1, for example, mature 23 S rRNA molecules all appear as a linear structure with none or a few internal loops (Klein et al., 1983). Instead, the rRNA molecules from strain ABLl look more complex. In 30 to 40% of all completely traceable molecules in three independent experiments, the short stem is the only feature of secondary structure remaining (as in the two molecules in Fig. l(a)), with the RNA chain held together in a large loop. In some other molecules, internal loops make the total contour difficult to trace; but at least 70% of the full-length molecules are circular, with a short stem visible in most of them (Fig. l(b)). Several precursor species were seen in the total RXA of the RNase III-deficient strain; and some, 0022%2836/85/230669-04
$03.00/O
but perhaps not all, of these speciesmust be active. To test this, the 5’ and 3’ termini of 23 S rRNA in 50 S ribosomes from the mutant were compared to those in polysomes. Figure 2 shows that the same termini were found in all the fractions examined. The 5’ end of 23 S rRNA in polysomes and 70 S and 50 S ribosomes from ABLl included species A, Cl and C2 (as described by King et al., 1984) up to 97 nucleotides longer than the mature terminus (lanes 2 to 4). Lane 1 shows the termini in the total RNA of the wild-type strain DlO (Gasteland, 1966). At the 3’ end (Fig. 2, lanes 6 to 8), two major species, A and C (see King et al., 1984), are up to 95 nucleotides longer than the published wild-type terminus (compare with lane 5) and are seen at comparable levels in 50 S ribosomes and in polysomes of the mutant. The stem at the base of the mutant rRNA has a AG” value of the order of - 100 kcal (1 kcal = 4.184 kJ), and a resulting stabilization much greater than other interactions in 23 S rRNA (Klein et al., 1983). Base-paired stems of intermediate stability can be formed in various regions of heated rRNA, and are stable even at 55°C in 70% formamide (Klein et aZ., 1985); the stem at the base of 23 S rRNA is thus even more stable. It therefore seemsvery likely that the 50 S ribosomes of the mutant strain function throughout the cycle of protein synthesis with the 5’ and 3’ termini of 23 S rRNA base-paired. This suggestion is strongest for the longest RNA chains; but because molecules with shorter stems are not disfavored in the population in polysomes, species with all possible lengths must be comparably functional. A short double-stranded stem (8 nucleotides long) has been inferred in models for the secondary structure of the mature 23 S rRNA (Noller et al., 1981), though its existence in ribosomes has not been verified and there is also no information about whether the putative stem might remain intact during protein synthesis. Our results make it increasingly likely that such a structure exists in wild-type ribosomes, though it is not stable enough to hold together in the conditions used for electron microscopy (Klein et al., 1983). The functionality of the 50 S ribosomes with long base-paired tails of
669
0
1985
Academic
Press
Inc.
(London)
Ltd.
670
R. Sirdeshmukh
and D. Schlessinger
Figure 1. Electron micrograph of a 23 S rRNA molecule from a 50 S ribosome of strain ABLl. 50 S ribosomes were dissolved in a 0.2% (w/v) sodium dodecyl sulfate, 10 mlcr-Tris. HCl (pH 7.4), 5 m&x-Mgcl,, and electrophoresed in a l”/b (w/v) agarose gel with Tris-acetate (pH 79). 5 mM-MgC1, as running buffer. The rRPu‘A was eluted from the gel slice in a buffer (Saha et aZ., 1983) containing 5 mM-Mgcl,, precipitated with cold ethanol, and dissolved and spread in 10 rnzxTris’ HCl (pH 7.4), 10 mM-EDTA, and 50% formamide (Klein et al., 1983). The 2 molecules labeled (a) are magnified 96,700 x The field at the right, labeled (b) is magnified 56,lOOO x ; the contour length of the molecule in (b) is the same (+O.l pm) as that in (a).
671
Letters to the Editor
1 Figure 2. Si nuclease analysis of the 5’ (left) and 3’ termini (right) of 23 S rRTu’A from strains DlO and ABLl’. Total R?jA from control strain DlO or RPU’A from polysomes or 70 S or 50 S ribosomes of strain ABLl was hybridized with single-stranded end-labeled DXA probes for the respective termini and S1 protection analysis assessed as described by King et al. (1984). The positions of the mature terminus M and the unprocessed species A, C, Cl and C2 are indicated (see King et al., 1984). Lanes 1 and 5, strain DlO RNA; lanes 2 and 6! ABLl polysomal RNA; lanes 3 and 7. RKA from ABLl 70 S ribosomes; lanes 4 and 8, RXA from ABLl 50 S ribosomes.
variable length in strain ABLl can therefore be rationalized if the termini of mature 23 S rRNA molecules are also base-paired and remain in close proximity in ribosomes during translation. The 3’ terminus of 23 S rRNA has been located at a point opposite the Ll binding site on the 50 S ribosome (Shatsky et al., 1980; Stoffler-Meilicke et al., 1981), and our results suggest that the 5’ terminus is located at the same point. Apparently any extra RNA sequences at this location block neither peptide
bond
formation
nor translocation.
We thank Grady Phillips and Tom Rucinsky for their extensive help in electron microscopy. These studies were supported in part by Kational Science Foundation grant PCM-8017402.
Ravi Sirdeshmukh David Schlessinger Department of Microbiology and Immunology Washington University School of Medicine St Louis, MO 63110, U.S.A. Received 14 May 1985, and in revised form 9 August 1985
References Bram, R. J., Young, R. A. & Steitz, J. A. (1980). Cell, 19, 393-401. Dunn, J. J. & Studier, F. W. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3296-3300. Gasteland, R. F. (1966). J. Mol. Biol. 16, 67-84. Kindler, P., Keil, T. & Hofschneider, P. (1973). Mol. Gen. Genet. 126, 53-69. King, T. C. & Schlessinger, D. (1984). J. Biol. Chem. 258, 12034-12042. King, T. C., Sirdeshmukh, R. & Schlessinger, D. (1984). Proc. Nat. Acad. Sci., U.S.A. 81, 185-188. Klein, B. K., King, T. C. & Schlessinger, D. (1983). J. Mol. Biol. 168, 809-830. Klein, B. K., Staden, A. & Schlessinger, D. (1985). Proc. Nat. Acud. Sci., U.S.A. 82, 3539-3542. Wikolaev, N., Silengo, L. & Schlessinger, D. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3361-3365. Pu’oller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, R. R., Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A., Gupta, R. & Woese, C. R. (1981). Nucl. Acids Rex 9, 6167-6189. Robertson, H. D., Webster, R. E. & Zinder. N. D. (1968). J. Biol. Chem. 243, 82-91. Saha, B., Strelow, S. t Schlessinger, D. (1983). J. B&hem. Biophys. Meth. 7, 277-284.
672
R. Sirdeshmukh and D. Schlessinger
Shatsky, I. pu’., Evstafieva, A. G., Bystrova, T. F.. Bogdanov, A. A. 6 Vasiliev, V. D. (1980). FEBS Letters, 122, 251-255. Sirdeshmukh, R. & Schlessinger, D. (1985). NucE. Acids Res. 13, 5041-5054.
Stoffler-Meilicke, M., Stoffler, G.. Odom, 0. W.. Zinn, 9.. Kramer, G. & Hardesty, B. (1981). Proc. Nat. Acad. Sci., U.S.A. 78, 5538-5542. Young, R. & St&z, J. (1978). Proc. LVat. Acad. Sci., U.S.A. 75, 3593-3597.
Edited by S. Brenner