Allele-specific structure probing of plasmid-derived 16S ribosomal RNA from Escherichia coli

Gene, 123 (1993) 75-80 0 1993 Elsevier Science Publishers

B.V. All rights reserved.

75

0378-l 119/93/$06.00

GENE 06866

Allele-specific structure from Escherichia coli (Ribosomes; extension)

rRNA functional

probing of plasmid-derived

sites; rRNA mutations;

site-directed

mutagenesis;

16s ribosomal

chemical

modification;

RNA

primer

Ted Powers and Harry F. Noller Sinsheimer Laboratories,

University of California, Santa Cruz, Santa Cruz, CA 95064, USA

Received by M. Belfort: 29 June 1992; Revised/Accepted:

20 August/21

August

1992; Received at publishers:

28 September

1992

SUMMARY

Biochemical analysis of Eschevichia coli ribosomes containing mutant 16s or 23s (r)ribosomal RNAs, produced via cloned rDNA genes on multicopy plasmids, has been hindered by the background of wild-type (wt) ribosomes containing host-derived rRNA. Here, we describe a method for the construction of unique priming sites in 16s rRNA that allow allele-specific structure probing of ribosomes containing plasmid-encoded RNA. Phenotypically silent mutations, designed to mimic related eubacterial sequences, have been introduced into four phylogenetically variable regions in the 16s rDNA gene that allow inspection of several 16s rRNA functional sites. When oligodeoxyribonucleotides complementary to these altered sequences are used to prime cDNA synthesis in primer extension reactions using reverse transcriptase, only plasmid-derived 16s rRNA is used as a template, thus rendering the wt background invisible. Unexpectedly, we were unable to introduce silent mutations into one nonconserved helix in 16s rRNA, suggesting that constraints in addition to Watson-Crick pairing are important in this region. -

INTRODUCTION

Characterization of rRNA mutations, produced from cloned rDNA genes on multi-copy plasmids in E. coli, has been aided by several recent advances. Genetic analysis of recessive mutations has been made possible by the discovery of selectable antibiotic-resistance markers in both 16s and 23s rRNA (Sigmund et al., 1984; Triman et al., 1989). These mutations can also be used to monitor the presence of plasmid-derived rRNA in r subunits, in 70s ribosomes, and in polysomes, using a quantitative Correspondence to: Dr. H.F. Noller, Sinsheimer Laboratories, UCSC, Santa Cruz, CA 95064, USA. Tel. (408) 459-2700; Fax (408) 459-3737. Abbreviations: A-site, r acceptor site; Ap, ampicillin; bp, base pair(s); cDNA, DNA complementary to RNA; kb, kilobase or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; P-site, r peptidyl site; pL, phage I major leftward promoter; r, ribosomal; R, resistance/resistant; rDNA, gene encoding rRNA; RDP, Ribosomal Database Project; Sp, spectinomycin; wt, wild type.

primer extension assay developed by Morgan and coworkers (Sigmund et al., 1987; Triman et al., 1988; Powers and Noller, 1990). In addition, use of the bacteriophage J. pL oL promoter/operator, in combination with the temperature-sensitive cI857-encoded repressor, allows the conditional expression of dominant lethal rRNA mutations (Gourse et al., 1985; Thomas et al., 1988; Vester and Garrett, 1988; Powers and Noller, 1990). Biochemical characterization of mutant ribosomes in vitro has remained hindered, however, by the fact that ribosomes isolated from cells are heterogeneous, containing both mutant and wt rRNA, the latter produced from seven host rRNA operons; this is most serious for mutations produced under the pL promoter, where the level of plasmid rRNA in ribosomes is at most 50% (Powers and Noller, 1990). One solution to this problem is to use synthetic rRNAs, generated by in vitro transcription of mutant 16s rRNA genes using T7 RNA polymerase, followed by in vitro reconstitution of 30s ribosomal su-

76

III

GAUU tttt

Fig. 1. Secondary structure schematic diagram of 165 rRNA (Gutell et al., 1985; Cute11 and Woese, 1990), showing allele-specific priming sites (boxed) used to study various I6S rRNA functional sites. Circled nt indicate positions altered by site-directed mutagenesis; mutations are in bold face.

bunits (Krzyzosiak et al., 1987; Melaqon et al., 1987). While this approach offers several attractive features, it also suffers distinct disadvantages, including the lack of post-transcriptionally modified bases in the RNAs, significantly lower functional activity of reconstituted subunits, and the inability to compare directly in vivo phenotypes with in vitro biochemical assays. Rapid chemical and enzymatic probing methods have

allowed the identification of residues in both 165 and 23s rRNA that interact with several functional ligands, including tRNA, elongation factors, and antibiotics (reviewed by Noller et al., 1990; Noller, 1991). This is most clearly demonstrated for tRNA, where specific residues in both 16s and 23s rRNA have been assigned as interacting with tRNA in the ribosomal A, P and E sites, as defined by conventional assays (Moazed and Noller,

77 TABLE

1

Construction Priming

of allele-specific

priming

Functional

siteb

sites in 16s rRNA”

siteC

II.

790 loop

III.

960 loop/900

IV.

1400 region

V.

1500 region

region

Organismd E. coli sym.Acy.psp E. coli Enteric eubact.”

Residues”

Sequence’

(1006-1022)

S-GCC-CUU-GAG-GCG-UGG-CU S-UUC-CAA-GAG-AAG-UGA-CU T-GUU-UUC-AGA-CU-GAG-AA

E. coli Am. calcoa’

(1450-1466)

S-GAA-UCC-AGA-GAU-GGA-UU T-UUC-GGG-AGG-GCG-CUU-AC

E. coli B. stearoj

(1514-1530)

(838-854)

5’-GCA-AAG-AGG-GCG-GUU-AC 5’-GGG-GAA-CCU-GCG-GUU-GG 5’-CCG-GAA-GGU-GCG-GCU-GG -

“Allele-specific priming sites were introduced into the 16s rRNA gene by site-directed mutagenesis, using the method of Kunkel (1985) modified use with phagemid vectors by Evnin and Craik (1988). Oligos used for mutagenesis are as follows: priming site II, 5-GAG-GTT-GTT-TCC-AAGAGA-AGT-GGC-TTC; priming site III, S-CAC-GGA-AGA-ATC-CAG-AGA-TGG-ATT-TGT-GCC-TTC; CCG-CAA-GGA-GGG-CG; priming site V, 5’-CCA-ACC-GCA-CCT-TCC-GGT-ACG-GTT-ACC. pSTLl02 (Triman et al., 1989) for phenotypic testing and molecular analysis, as described in section

priming site IV, 5’-GCT-TAAThese mutations were introduced into b. Construction of priming site III has been

described previously (Powers and Noller, 1991). b Priming site numbers correspond to the regions in 16s rRNA denoted in Fig. 1. ‘Functional sites monitored by each priming site are described briefly in section a and are reviewed in detail in Noller dOrganisms

refer to eubacterial

species obtained

from the RDP or R. Gutell

(personal

communication),

for

et al. (1990) and Noller (1991).

as indicated.

“The nt numbers correspond to the sequence of E. coli 16s rRNA. ‘Different eubacterial sequences are indicated below that of E. coli; changes introduced into the E. coli 16s rRNA gene are underlined. Oligos complementary to the lower sequence are used for priming cDNA synthesis from plasmid-derived 16s rRNA; oligos complementary to the E. coli sequence can be used for priming cDNA synthesis from chromosomally derived 16s rRNA. pStr. symbiont S of Acyrthosyphon pisum (purple gamma division) (RDP). “Enteric eubacterium (purple gamma division) (R.R. Gutell, personal communication). ‘Acinetobacter calcoaceticus ‘Bacillus stearothermophilus

(purple gamma division) (RDP). (Gram+ eubacteria) (RDP).

1989; 1990). Thus, in many cases it is possible to monitor the functional state of the ribosome by inspection of the pattern of modification of the RNAs. Detection of modified nts is accomplished by a primer extension assay that uses oligos to prime cDNA synthesis from specific positions in the RNA. By introducing phenotypically silent mutations into phylogenetically variable regions in the plasmid-derived rRNAs, we can selectively probe the mutant ribosomes, in spite of the presence of ribosomes containing wt, host-derived rRNA (Douthwaite et al., 1989; Powers and Noller, 1991). The aim of the present study was to construct several such allele-specific priming sites in 16s rRNA.

EXPERIMENTAL

AND DISCUSSION

(a) Design of allele-specific priming sites in 16s rRNA Four unique priming sites were introduced into 16s rRNA by site-directed mutagenesis (Fig. 1; Table I). These mutations, referred to as priming sites II through V, alter the sequence of 16s rRNA without changing its predicted secondary structure or affecting its function. Priming sites II, III, and V alter the sequence of 16s rRNA within helical regions while priming site IV involves swapping one common tetraloop motif for another (Woese et al., 1990).

The locations of these sites were chosen with two priorities in mind. First, to reduce the probability of introducing deleterious mutations, changes were made in regions of the RNA that are phylogenetically variable and, furthermore, mimic sequences found in closely related eubacterial 16s rRNA sequences (Woese, 1987) (Table I). The most divergent organism used, relative to E. coli, was the Gram+ Bacillus stearothevmophilus, for priming site V; other purple eubacterial species were used for priming sites IIIIV (Table I). All sequences used for the design of these priming sites were from the RDP (Olsen et al., 1991), except for the sequence of priming site III, which was obtained from R. Gutell (personal communication). Second, the priming sites have been placed downstream from highly conserved regions in 16s rRNA that are strongly implicated in r function (reviewed by Noller et al., 1990; Noller, 1991). Priming site II allows probing of the 690 region and the 790 loop in 16s rRNA, which contain residues footprinted by P-site tRNA (Moazed and Noller, 1990). Priming site III allows probing of nt in the 960 loop and position G926, which are also footprinted by P-sitebound tRNA. In addition, this priming site allows inspection of the conserved 900 region, which is involved in interactions with streptomycin and tetracycline (Moazed and Noller, 1987). Priming sites IV and V allow probing of the universally conserved 1400 and 1500 regions,

78

-G791

-G1417 -G1494

-3mU1498

Fig. 2. Autoradiograph demonstrating the specificity of oligo primers for 16s rRNA containing allele-specific priming site mutations. For each set of samples in A-D, 16s rRNAs contain (+) or lack (-) one of the allele-specific priming sites II-V, as indicated at the top. G and C are dideoxy sequencing lanes and refer to the sequence of 16s rRNA; K indicates sequencing reactions without ddNTPs. For reference, nt within 16s rRNA functional sites are indicated on the right. 16s rRNA was prepared as described by Powers and Noller (1991) from E. coli cells transformed with either the wt control plasmid, pSTL102, or with derivatives of pSTL102 carrying one of the specific priming sites. Primer extension reactions were performed as described (Stern et al., 1988) except that hybridization, extension, and chase reactions were carried out at 50°C when using the priming site V-specific oligo. Each reaction contained a total of 0.25 ,ng of 16s rRNA. Samples were run on a 6% polyacrylamide sequencing gel (1:20 bis:acrylamide), as described (Stern et al., 1988). Oligo primers are 17mers complementary to the sequences listed in Table I. Primers were used as dilutions of 1 ,ug/50 ~1 stocks, as follows: priming sites II and III, 1:50; priming site IV, 1:200; priming site V, 1:lO. At higher concentrations, some of these oligos were found to prime cDNA synthesis containing wt control RNA samples.

at other locations

in the RNA, giving rise to a background

respectively. These regions are located at the site of codon-anticodon interaction and contain bases footprinted by A- and P-site-bound tRNA, as well as by several miscoding antibiotics (Moazed and Noller, 1987; 1990). (b) Characterization of priming sites The priming site mutations were introduced into pSTL102 (Triman et al., 1989) for phenotypic testing. This plasmid contains an ApR gene and the entire rrnB operon from E. coli. Here, the rDNA genes are expressed from the strong, constitutive P,P, promoters, contributing approx. 70% of the 16s and 23s rRNA incorporated into ribosomes (Sigmund et al., 1987; Triman et al., 1988). In addition, a C+U transition at position 1192 in the 16s rRNA gene confers resistance to Sp (Sigmund et al., 1984) allowing for the direct phenotypic characterization of mutant 16s rRNA genes. Derivatives of pSTL102, each carrying one of the priming sites, were used to transform E. coli strain DHl (Triman et al., 1989). These mutations were found to be completely phenotypically silent, as judged by the normal growth of cells on selective agar plates containing either 100 pg Ap/ml or both 100 pg Ap/ml and 50 pg

of primer

extension

products

in samples

Sp/ml, at each of three temperatures tested, 30°C 37°C and 42°C (data not shown). In addition, these alterations did not affect the amount of plasmid-encoded 16s rRNA incorporated in ribosomes, determined by a quantitative primer extension assay (Sigmund et al., 1987; data not shown). The specificity of oligos for priming cDNA synthesis from 16s rRNA containing the priming site alterations is shown in Fig. 2. We have demonstrated previously the efficacy of priming site III (Powers and Noller, 1991). (c) Priming site I We have so far been unsuccessful at introducing a priming site in 16s rRNA that allows inspection of the universally conserved 530 loop region 16s rRNA (Fig. 1). The 530 loop is thought to play an essential role in the binding of tRNA to the A-site (reviewed by Noller, 1991) and the examination of mutations in this region may prove essential to an understanding of its precise function. In three attempts, mutations introduced into nonconserved portions of the 620 helix in 16s rRNA produced deleterious phenotypes when expressed in pSTL102; cells trans-

79 Growth

Oraanism

On Spectinomych

4O

AACUGCAUCUGA E. co/i

l ~IIIlllI GUGUAG+CU

ij20-CAAEYE UCGGGCCC

?i

C. jejuni

+++

+**

600

C,UAAA

610

620-E

**+

A6i:UGcAUCUOA

AACCWG I I * I I I CGGGCCC

.*lIllllI OUGUAG+CU

‘PAA~

600

610

UGCAUCU5A .*fllllll

UA

UGCAIJCUGA *

*

++

+I-

+

*+

+I-

+

I-*

UA

B.

UGCAUCUGA

stearo

~.~ara~a~~ +

P. vulgaris

u+-

UC . .

630

; t

AAC

ACQU@3G II.llI cMw+x~ 4

i

UGCAUCUGA ..11ll!ll GUGUAGqCU

‘UAAA

600

AG

Fig. 3. Summary of attempts to construct an allele-specific priming site in the 620 helix of 16s rRNA. Alterations were modeled after other eubacteria, as indicated, and introduced into the 16s rRNA gene by site-directed mutagenesis; the alterations indicated as V. parnkaem and P. vulgaris are a composite of sequences found in these two closely related organisms. The mutations were expressed via pSTLlO2 in E. coii strain DIII and the resulting phenotypes were tested on agar plates containing 100 pg Spjml at 3O”C, 37”C, and 42”C, as shown. Relative colony sizes are as follows: + + +, large, wt-size colonies; + + , medium-sized colonies; + , small-sized colonies; + / -, weak growth only (no isolated colonies); - , no growth. Abbreviations: C. jejuni, Cu~~~y~o~uc~er jejuni (purple delta division of eubacteria); B. steam, Bacifhs steu~otk~r~~p~?i~~s (Gram’ eubacteria); V. parahaem, Vibrio ~ur~~erno~~~~c~s (purple gamma division of eubacteria); P. umlgaris, Proteus vulgaris (purple gamma division).

formed with the mutant plasmids grew normally on Ap plates but produced cold-sensitive phenotypes on Spcontaining plates (Fig. 3). For one of the organisms mimicked, C. jej~n~, we fortuitously obtained two sets of mismatch mutations during the mutagenesis procedure. When expressed in pSTL102, these mutations were completely nonviable on Sp {Fig. 3). These results suggest that

constraints in addition to the simple requirement for Watson-Crick pairing are important in this helix and indicate that despite their lack of phylogenetic conservation, certain regions in rRNA are not amenable to this mutagenesis procedure. Reasons for this unanticipated inflexibility may include interactions with r-proteins or fortuitous base pairing elsewhere in the RNA.

80 (d) Conclusions The method described in this report for the allele-specific structure probing of plasmid-encoded 16s rRNA opens the door for the biochemical characterization of ribosomes produced in E. coli carrying a variety of 16s rRNA mutations. Indeed, we have demonstrated the usefulness of this approach recently to study the interaction between ribosomes carrying streptomycin-resistance mutations in 16s rRNA and streptomycin, the antibiotic

using the footprint

afforded

by

in the 900 region of 16s rRNA as an assay

for drug binding (Powers and Noller, 199 1). More recently, this method has allowed us to analyze the functional lesion in ribosomes carrying a dominant lethal mutation at position G530 in 16s rRNA, by monitoring the reactivities of bases in 16s rRNA footprinted by tRNA (T.P. and H.F.N., manuscript submitted). The feasibility of this method for the study of 23s rRNA mutations has been established as well (Douthwaite et al., 1989; Aagaard et al., 1991). Our difficulty in introducing silent mutations into the 620 helix of 16s rRNA should be taken as a warning, however, that some non-conserved regions in ribosomal RNA may be refractory to this approach, emphasizing the need to examine rigorously the phenotypes of any new priming site mutations. While this method has so far been used solely in in vitro assays, the permeability of E. coli cells to several chemical probes, including kethoxal and dimethylsulfate, should allow specific structure probing of mutant ribosomes in vivo as well.

ACKNOWLEDGEMENTS

We are grateful to M. Ares for discussions that led to the development of the allele-specific probing method presented in this paper. We also thank R. Gutell for helpful discussions and for providing 16s rRNA sequence alignments. This work was supported by NIH grant GM17129 and NSF grant DMB-8704076 (to H.F.N.) and a Dissertation Year Fellowship from the University of California (to T.P.).

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E.A.: Antibiotic

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