Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.

Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction.     

Location and characteristics of ribosomal protein binding sites in the 16S RNA of Escherichia coli.

Specific binding sites for five proteins of the Escherichia coli 30S ribosomal subunit have been located within the 16S RNA. The sites are structurally diverse and range in size from 40 to 500 nucleotides; their functional integrity appears to depend upon both the secondary structure and conformation of the RNA molecule. Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly.     

The characterisation of a fragment of ribosomal protein S4 that is protected against trypsin digestion by 16S ribosomal RNA of Escherichia coli.

After mild trypsin treatment of a complex of ribosomal protein S4 and 16S RNA of Escherichia coli, a large homogeneous fragment of the S4 protein was protected against digestion by its RNA binding site. This fragment was isolated and characterised for molecular weight. It was able to rebind specifically to 16S RNA. Preliminary results indicate that protected protein fragments can also be obtained from other proteins that complex specifically with 23S and 5S RNA.     

Secondary structure of a 345-base RNA fragment covering the S8/S15 protein binding domain of Escherichia coli 16S ribosomal RNA

A technique for isolating defined fragments of a large RNA has been developed and applied to a ribosomal RNA. A section of the Escherichia coli rrnB cistron corresponding to the S8/S15 protein binding domain of 16S ribosomal RNA was cloned into a single-stranded DNA phage; after hybridization of the phage DNA with 16S RNA and digestion with T1 ribonuclease, the protected RNA was separated from the DNA under denaturing conditions to yield a 345-base RNA fragment with unique ends (bases 525-869 in the 16S sequence). The secondary structure of this fragment was determined by mapping the cleavage sites of enzymes specific for single-stranded or double-helical RNA. The fragment structure is almost identical with that proposed for the corresponding region of intact 16S RNA on the basis of phylogenetic comparisons [Woese, C. R., Gutell, R., Gupta, R., & Noller, H. (1983) Microbiol. Rev. 47, 621-669]. We conclude that this section of RNA constitutes an independently folding domain that may be studied in isolation from the rest of the 16S RNA. The structure mapping experiments have indicated several interesting features in the RNA structure. (i) The largest bulge loop in the molecule (20 bases) contains specific tertiary structure. (ii) A region of long-range secondary structure, pairing bases about 200 residues apart in the sequence, can hydrogen bond in two different mutually exclusive schemes. Both appear to exist simultaneously in the RNA fragment under our conditions. (iii) The long-range secondary structure and one adjacent helix melt between 37 and 60 degrees C in the absence of Mg2+, while the rest of the structure is quite stable.     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Mutations in the 915 region of Escherichia coli 16S ribosomal RNA reduce the binding of streptomycin to the ribosome.

The nine possible single-base substitutions were produced at positions 913 to 915 of the 16S ribosomal RNA of Escherichia coli, a region known to be protected by streptomycin [Moazed, D. and Noller, H.F. (1987) Nature, 327, 389-394]. When the mutations were introduced into the expression vector pKK3535, only two of them (913A----G and 915A----G) permitted recovery of viable transformants. Ribosomes were isolated from the transformed bacteria and were assayed for their response to streptomycin in poly(U)- and MS2 RNA-directed assays. They were resistant to the stimulation of misreading and to the inhibition of protein synthesis by streptomycin, and this correlated with a decreased binding of the drug. These results therefore demonstrate that, in line with the footprinting studies of Moazed and Noller, mutations in the 915 region alter the interaction between the ribosome and streptomycin.     

A method of preparing Escherichia coli 16 S RNA possessing previously unobserved 30 S ribosomal protein binding sites

A method of preparing 16 S RNA has been developed which yields RNA capable of binding specifically at least 12, and possibly 13, 30 S ribosomal proteins. This RNA, prepared by precipitation from 30 S subunits using a mixture of acetic acid and urea, is able to form stable complexes with proteins S3, S5, S9, S12, S13, S18 and possibly S11. In addition, this RNA has not been impaired in its capacity to interact with proteins S4, S7, S8, S15, S17 and S20, which are proteins that most other workers have shown to bind RNA prepared by the traditional phenol extraction procedure (Held et al., 1974; Garrett et al., 1971; Schaup et al., 1970,1971).We have applied several criteria of specificity to the binding of proteins to 16 S RNA prepared by the acetic acid-urea method. First, the new set of proteins interacts only with acetic acid-urea 16 S RNA and not with 16 S RNA prepared by the phenol method or with 23 S RNA prepared by the acetic acid-urea procedure. Second, 50 S ribosomal proteins do not interact with acetic acidurea 16 S RNA but do bind to 23 S RNA. Third, in the case of protein S9, we have shown that the bound protein co-sediments with acetic acid-urea 16 S RNA in a sucrose gradient. Additionally, a saturation binding experiment showed that approximately one mole of protein S9 binds acetic acid-urea 16 S RNA at saturation. Thus, we conclude that the method employed for the preparation of 16 S RNA greatly influences the ability of the RNA to form specific protein complexes. The significance of these results is discussed with regard to the in vitro assembly sequence.     

A conformational switch involving the 915 region of Escherichia coli 16 S ribosomal RNA

A novel alternative conformation, which involves an interaction between the 5' terminal and 915 regions (E. coli numbering), is proposed after a screening of compiled sequences of small subunit ribosomal RNAs. This conformation contains a pseudoknot helix between residues 12-16 and 911-915, and its formation requires the partial melting of the 5' terminal helix and the disruption of the 17-19/916-918 pseudoknot helix of the classical 16 S rRNA secondary structure. The alternate pseudoknot helix is proximal to the binding site of streptomycin and various mutations in rRNA which confer resistance to streptomycin have been located in each strand of the proposed helix. It is suggested that the presence of streptomycin favours the shift towards the alternate conformation, thereby stabilizing drug binding. Mutations which destabilize the novel pseudoknot helix would restrict the response to streptomycin.     

Primary sequence of the 16S ribosomal RNA of Escherichia coli.

Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E. coli is described. The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e. about 95% of the molecule. Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented. In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.     

Chromosomal loci for 16S ribosomal RNA in Escherichia coli

Summary Genetic loci for 16S ribosomal RNA (rRNA) on the Escherichia coli chromosome were determined using the K-sequence, a characteristic oligonucleotide of strain K12, as a genetic marker. Oligonucleotide analyses of 16S rRNA from various recombinants between strain K12 and strain B(H) showed that the loci for 16S rRNA containing the K-sequence were near the metB locus which was at 77 min. on the chromosome map.     

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of 'domain IV' of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757-1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2-protected oligonucleotides, also derived from domain IV (i.e. from residues 1955-2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.     

Refined secondary structure models for the 16S and 23S ribosomal RNA of Escherichia coli

The complete range of published sequences for ribosomal RNA (or rDNA), totalling well over 50,000 bases, has been used to derive refined models for the secondary structures of both 16S and 23S RNA from E. coli. Particular attention has been paid to resolving the differences between the various published secondary structures for these molecules. The structures are described in terms of 133 helical regions (45 for 16S RNA and 88 for 23S RNA). Of these, approximately 20 are still tentative or unconfirmed. A further 20 represent helical regions which definitely exist, but where the detailed base-pairing is still open to discussion. Over 90 of the helical regions are however now precisely established, at least to within one or two base pairs.