The 3' terminal colicin fragment of Escherichia coli 16S ribosomal RNA. Conformational details revealed by enzymic and chemical probing

The conformation of the colicin fragment of E. coli 16S rRNA was probed with various nucleases and with the adenosine-specific reagent diethylpyrocarbonate (DEP). The results confirm the presence of a stable central hairpin in the colicin fragment and a weaker additional secondary structure involving the regions 5' and 3' to this hairpin. By monitoring DEP accessibility at various stages of heat-denaturation sequential unfolding of individual base pairs was followed. In agreement with previous results it could be shown that dimethylation of the two adjacent adenosines in the hairpin loop (a feature in virtually all ribosomes) leads to a destabilization of the hairpin helix. Accessibilities of G residues, involved in the weaker additional secondary structure is anomalous. One G residue is sensitive to the single strand specific RNase T1 and insensitive to DEP, while the situation is reversed for the adjoining G residue. The strong reaction of the latter G-residue with DEP is unusual and indicates a very special conformation.     

Regulatory state of ribosomal genes and physiological changes in the concentration of free ribonucleic acid polymerase in Escherichia coli.

The concept of promoter efficiency is introduced as frequency of RNA chain initiation at a given promoter normalized to the intracellular concentration of free (but functional) RNA polymerase. Previous observations from this laboratory on the synthesis of ribosomes and beta-galactosidase are used to show that during a nutritional shift-up from succinate minimal to glucose-amino acids medium (3-fold increase in steady-state growth rate) the concentration of free (active) RNA polymerase decreases to one-quarter of the pre-shift value and the promoter efficiencies of the genes for ribosomal RNA and ribosomal proteins increase 9- and 6-fold respectively. This extent of control of ribosomal genes is much greater than expected on the basis of the increase in the rate of ribosome synthesis (3-fold).     

Biogenesis of ribosomes: free ribosomal protein pools in Escherichia coli

Proteins from ribosomal subunits (30 s and 50 s) have been fractionated into split (SP-30 and SP-50) and core (core-30 and eore-50) proteins. Antisera prepared in rabbit against them are shown to be highly group-specific as judged by the Ouchterlony (1967) double diffusion test and by precipitin reaction in solution. Various parameters which influence the immuno-precipitation of these proteins by specific antisera have been investigated. It is demonstrated that under controlled conditions this provides a sensitive and reliable method for characterization and quantitative estimation of free ribosomal proteins. The technique has been successfully applied in the investigations of various properties of free ribosomal protein pools existing in Escherichia coli. It is concluded that free ribosomal proteins in E. coli may constitute 8 to 14% of total soluble proteins under different growth conditions. Relative pool sizes of four classes of proteins expressed as a percentage of the total soluble proteins in bacteria grown in L broth (doubling period, 30 min) are estimated to be core-30, 2.1; core-50, 3.4; SP-30, 3.6; SP-50, 5.0. From the studies on bacteria grown in different media (doubling period, 30, 42 and 60 min), we further conclude that the amount of free proteins increases with the growth rate so as to constitute a constant fraction (7 to 9%) of total ribosomal proteins. Relative pool-sizes corresponding to four classes of ribosomal proteins, however, remain unaltered by different growth rate.     

Identification and characterization of E.coli ribosomal binding sites by free energy computation.

Sequences upstream from translational initiation sites of different E.coli genes show various degrees of complementarity to the Shine-Dalgarno (SD) sequence at the 3' end of the 16S rRNA. We propose a quantitative measure for the SD region on the mRNA, that reflects its degree of complementarity to the rRNA. This measure is based on the stability of the rRNA-mRNA duplex as established by free energy computations. The free energy calculations are based on the same principles that are used for folding a single RNA molecule, and are executed by similar algorithms. Bulges and internal loops in the rRNA and mRNA are allowed. The mRNA string with maximum free energy gain upon binding to the rRNA is selected as the most favorable SD sequence of a gene. The free energy value that represents the SD region provides a quantitative measure that can be used for comparing SD sequences of different genes. The distribution of this measure in more than 1000 E.coli genes is presented and discussed.     

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)     

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.     

Acetylation of L12 increases interactions in the Escherichia coli ribosomal stalk complex

The ribosomal stalk complex in Escherichia coli consists of L10 and four copies of L7/L12, and is largely responsible for binding and recruiting translation factors. Structural characterisation of this stalk complex is difficult, primarily due to its dynamics. Here, we apply mass spectrometry to follow post-translational modifications and their effect on structural changes of the stalk proteins on intact ribosomes. Our results show that increased acetylation of L12 occurs during the stationary phase on ribosomes harvested from cells grown under optimal conditions. For cells grown in minimal medium, L12 acetylation and processing is altered, resulting in deficient removal of N-terminal methionine in ∼ 50% of the L12 population, while processed L12 is almost 100% acetylated. Our results show also that N-acetylation of L12 correlates with an increased stability of the stalk complex in the gas phase. To investigate further the basis of this increased stability, we applied a solution phase hydrogen deuterium exchange protocol to compare the rate of deuterium incorporation in the proteins L9, L10, L11 and L12 as well as the acetylated form of L12 (L7), in situ on the ribosome. Results show that deuterium incorporation is consistently slower for L7 relative to L12 and for L10 when L7 is predominant. Our results imply a tightening of the interaction between L7 and L10 relative to that between L12 and L10. Since acetylation is predominant when cells are grown in minimal medium, we propose that these modifications form part of the cell's strategy to increase stability of the stalk complex under conditions of stress. More generally, our results demonstrate that it is possible to discern the influence of a 42 Da post-translational modification by mass spectrometry and to record subtle changes in hydrogen/deuterium exchange within the context of an intact 2.5 MDa particle.     

Identification of form III conformers in tRNAPhe from Escherichia coli by intramolecular photo-cross-linking

In the absence of divalent cations, at neutral pH, low ionic strength, and low to moderate temperature, tRNAs are known to be in a denatured form, designated form III in the tRNA phase diagram by Cole et al. [Cole, P. E., Yang, S. R., & Crothers, D. M. (1972) Biochemistry 11, 4358-4368]. Form III tRNAPhe from Escherichia coli has been studied at pH 7, 5 mM Na+, and 10 degrees C. As judged from ethidium bromide intercalation, it exhibits extensive secondary structure. 4-Thiouridine in position 8 of the tRNAPhe sequence was used as a built-in photoaffinity probe. Spectroscopic and spectrofluorometric analysis in the near-UV range of form III tRNAPhe irradiated with broad-band near-UV light to completion of the reaction before or after reduction with NaBH4 revealed that the Pdo(4-5)Cyt (8-C) and Pdo(4-5)Urd (8-U) adducts form in equimolar yield. In different experiments, the overall yield of s4U conversion to these adducts varies between 20 and 40%. The remaining s4U is photolyzed to weakly absorbing product(s) in the near-UV range. The disappearance of s4U follows biexponential kinetics while the 8-C adduct formation follows monoexponential kinetics, indicating the presence of at least two tRNA classes of conformers, not in equilibrium on the time scale of the reaction. Migration on a denaturing polyacrylamide gel of irradiated form III tRNAPhe revealed three main bands, D1, D2, and D3, and no slowly migrating tRNA dimers. D1 migrates at the control position and presumably contains the photolysis product(s) P. The fast-migrating D2 and D3 bands contain 8-Pyr cross-links which were identified by sequence analysis as 8-(66-68) in D2 and 8-(40-43) and 8-(59-62) in D3. On the basis of these data, it is proposed that the minor poorly photoreactive class II conformers are the cloverleaf and close variants, whereas the major class I cross-linkable conformers are essentially long-extended secondary structures. Clearly, our data demonstrate the polymorphism of form III tRNAPhe.     

Orientations of transfer RNA in the ribosomal A and P sites.

In protein synthesis, peptide bond formation requires that the tRNA carrying the amino acid (A site tRNA) contact the tRNA carrying the growing peptide chain (P site tRNA) at their 3' termini. Two models have been proposed for the orientations of two tRNAs as they would be bound to the mRNA in the ribosome. Viewing the tRNA as an upside down L, anticodon loop pointing down, acceptor stem pointing right, and calling this the front view, the R (Rich) model would have the back of the P site tRNA facing the front of the A site tRNA. In the S (Sundaralingam) model the front of the P site tRNA faces the back of the A site tRNA. Models of two tRNAs bound to mRNA as they would be positioned in the ribosomal A and P sites have been created using MC-SYM, a constraint satisfaction search program designed to build nucleic acid structures. The models incorporate information from fluorescence energy transfer experiments and chemical crosslinks. The models that best answer the constraints are of the S variety, with no R conformations produced consistent with the constraints.     

Topography of the Escherichia coli ribosomal 30S subunit-initiation factor 2 complex.

The specific effect of the binding of initiation factor IF2 on E coli 16S rRNA within the [IF2/30S/GTP] complex has been probed by crosslinking experiment with trans-diamminedichloro platinum (II) and by phosphate alkylation with ethylnitrosourea. Several 16S rRNA fragments crosslinked to IF2 have been identified and are mostly located in the head and the lateral protrusion of the 30S subunit. The study of the effect of IF2 binding to the 30S subunit reveals that the factor does not tightly bind to the 16S rRNA and induces both isolated reductions and enhancements of phosphate reactivity in the 16S rRNA. Several of them are located near the binding site of IF2 and weak effects are observed in distant parts of the subunit. These results are discussed in the light of current knowledge of the topographical localization of IF2 with the 30S subunit and of its relation with function.     

A new ribosomal mutation which affects the two ribosomal subunits in Escherichia coli.

Summary The nif cistrons indentified by complementation analysis in the preceding paper (Dixon et al., 1977) were mapped with respect to hisD and to each other by Pl cotransduction and three-factor reciprocal crosses. The order obtained was hisD nifB nifA (nifL) nifF nifE nifK nifD nifH. Analysis of hisD2-nif cotransduction data by the Wu equation (Wu, 1966) suggested that the nif genes are divided into two clusters: a his-proximal cluster comprising nifBA(L)F and a his-distal group of nifEKDH.     

Study on conformational states of Escherichia coli tRNAPhe in solution by a modulation-free ESR-spectrometer

A modulation free Electron Spin Resonance spectrometer was used for the registration of spectral absorption lines of a spin-labeled Escherichia coli phenylalanine tRNA in solution with low (less than 0.1%) line shape distortion. The analysis of line shape of two different spin-labels introduced into position 8 revealed that phenylalanine tRNA in solution exists as a mixture of two conformers, the equilibria between conformers being dependent on pH, concentration of magnesium and functional state of tRNA (deacylated, aminoacylated or peptidylated). There are no overall structural rearrangements upon aminoacylation or peptidylation of tRNA. The observed small changes of spectral line shape can be assigned to shifts in conformational equilibria.     

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.     

Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe.

A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.     

Secondary structure as primary determinant of the efficiency of ribosomal binding sites in Escherichia coli.

Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E. coli and then studied the variation in expression of the fusions. The RBSs originated from bacteriophage Q beta and MS2 genes and the E. coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK). The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold. Lac-specific mRNA synthesis follows the variation in protein production. It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon. This efficiency is context dependent. The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results. Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.     

Structural behavior of RNA-binding proteins in free and complex states: ribosomal protein L25 and 5S RNA from Escherichia coli

A novel approach has been proposed to evaluate the steadiness of polar clusters containing RNA-binding sites on the protein surface. The degree of clusterization of RNA-binding polar residues can be a measure of the steadiness of corresponding polar clusters. Ribosomal protein L25 from E. coli forms a complex with a fragment of 5S rRNA by means of two binding sites S1 and S2. We have examined cluster distribution of RNA-contacting polar residues on the protein surface by using the data of two states: complex state (in crystal and solution) and free state (in solution). For the crystal, the extent of clusterization of binding sites S1 and S2 are estimated to be 74.1 and 100%, respectively. For the free state in solution, the degrees of clusterization of these two sites are 22.8 and 68.6%, respectively. Thus, we have obtained a steadiness quantitative measure of two different types of protein sites for binding to RNA: one for the already existing protein binding site, and the other for the RNA-induced protein binding site. It was shown that definite variations of the protein structure in crystal and in solution can be of significant functional meaning. The result could be applied to the structural behavior of numerous protein complexes with double-stranded RNA and DNA.