Structural and functional studies of ribonucleoprotein fragments isolated from Escherichia coli 50 S ribosomal subunits.

Ribonuclease digestion of 50 S-derived LiCl cores led to 22 ribonucleoprotein particles which were isolated by repeated sucrose gradient centrifugations. The protein content was determined and ranged from 2 to 28 proteins. Most of the fragments showed a unique RNA pattern as judged by acrylamide gel electrophoresis.Functional tests were performed with selected fragments. No fragment was active in the poly(U) or the peptidyl-transferase assay. Chloramphenicol binding studies revealed that in addition to the dominant role of protein L16, the protein L11 (or L6) is involved directly in the drug binding. Finally, tests for ATPase and GTPase activity showed that protein L18 is involved in GTPase activity.     

Localization of the 3' end of Escherichia coli 16 S RNA by electron microscopy of antibody-labelled subunits.

The 3′ end of 16 S RNA is localized on the 30 S subunit of Escherichia coli ribosomes by immune electron microscopy. It is located in the groove between the side “ledge” and the “head” of the subunit on the level of the ledge top. Thus, we have localized the 30 S subunit functional site which is believed to be responsible for binding of the specific messenger RNA sequence preceding the initiation codon. The localization of the 3′ end of 16 S RNA has been done by a new approach in immune electron microscopy. It is based on the covalent binding of low molecular weight ligands, containing the residue of phenyl-β-d-lactoside hapten, to certain points of RNA and the localization of the binding site of the antibody specific to this hapten by electron microscopy. The advantages of this approach in comparison with conventional methods of immune electron microscopy are discussed.     

Modification of specific lysine residues in E. coli methionyl-tRNA synthetase by crosslinking to E. coli formylmethionine tRNA

A protein affinity labeling derivative of E. coli tRNAfMet has been prepared which carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond capable of reaction with cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine epsilon-amino groups in proteins. Reaction of the modified tRNA with E. coli methionyl-tRNA synthetase leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence. Digestion of the crosslinked enzyme with trypsin followed by peptide mapping reveals that the major crosslinking reactions occur at four specific lysine residues, with minor reaction at two additional sites. Native methionyl-tRNA synthetase contains 90 lysine residues, 45 in unique sequences of the dimeric alpha 2 enzyme. Crosslinking of the protein to different regions in tRNAfMet thus occurs with the high degree of selectivity necessary for use in determining the peptide sequences which are near specific nucleotide sequences of tRNA bound to the protein.     

Identification of protein S 1 at the messenger RNA binding site of the Escherichia coli ribosome

Poly-4-thiouridylic acid acts as messenger RNA for polyphenylalanine synthesis in an $\text{in vitro}$ protein synthesizing system. When a complex consisting of ribosomes, poly-4-thiouridylic acid and Phe-tRNA is irradiated at 300 to 400 nm, covalent bonds between this messenger RNA and protein S 1 are formed.     

Study of the internal structure of Escherichia coli ribosomes by neutron and x-ray scattering.

Neutron scattering curves of the small and large subparticles of Escherichia coli ribosomes are presented over a wide range of scattering angles and for several contrasts. It was verified that the native ribosome structure was not affected by ²H₂O in the buffer. The reliability of the neutron scattering curves, obtained in H₂O buffer, was established by X-ray scattering experiments on the same material.The non-homogeneous distribution of RNA and protein in the subparticles of E. coli ribosomes is confirmed, with RNA predominantly within the particle and protein predominantly on its periphery. The distances between the centres of gravity of the RNA and protein components do not exceed 25 Å and 30 Å, in the large and small subparticles, respectively.The volume occupied by the RNA within the large and small subparticles is determined. The ratio of the “dry” volume of the RNA to the occupied volume is found to be 0.56; it is the same in both subparticles. Such packing of RNA is characteristic of single helices of ribosomal RNA at their crystallization and of the helices in transfer RNA crystals. A conclusion is drawn that RNA in ribosomes is in a similar state.Experimental scattering curves for the small subparticle depend significantly on the contrast in the angular region in which the scattering is mainly determined by the particle shape. The scattering curve, as infinite contrast is approached, is similar to that calculated for the particle as observed by electron microscopy. Thus, the long-existing contradiction between electron microscopy data (an elonggated particle with an axial ratio 2:1) and X-ray data (an oblate particle with an axial ratio 1:3.5), concerning the overall shape of the 30 S subparticle, is settled in favour of electron microscopy. The experimental neutron scattering curve of RNA within the small subparticle is well-described by the V-like RNA model proposed recently by Vasiliev et al. (1978).Experimental data are given to support the hypothesis that the maxima in the X-ray scattering curves, in the region of scattering angles corresponding to Bragg distances of 90 to 20 Å, arise from the ribosomal RNA component alone. It is shown that the prominence of the peaks in this region of the scattering curve depends only on the scattering fraction of the RNA component. The scattering fraction can be changed both by using the “native contrast” (ribosomal particles containing different amounts of protein) and by varying the solvent composition. The maxima are most pronounced where the RNA scattering fraction is highest or in solvents where the protein density is matched by the solvent. The scattering vectors of the maxima in the X-ray and neutron scattering curves, however, remain unchanged. This allows us to propose the tight packing of RNA as a common principle for the structural arrangement of RNA in ribosomes.     

Rotational diffusion of Escherichia coli ribosomes. II. - Ribosome attachment to polyurydilic acid.

Ribosome attachment to poly(U) has been studied by following the rotational diffusion of polyribosomes in solution. On the average, 13-17 and 50 nucleotides are found to be associated with 30S and 70S ribosome respectively. For an equal length of poly(U), the number of particles in a 30S polysome is four times that in a 70S polysome. The results are consistent with a structure of the polysome in which individual ribosomes are in close contact.     

Ribosomal components required for binding protein S1 to the 30 S subunit of Escherichia coli.

30 S subunits of Escherichia coli ribosomes washed with 3 m-NH₄C1 lose proteins S2, S3, S9, S10, S14, S20 and S21, as well as their ability to bind S1 with high affinity (Laughrea and Moore, 1978). Binding activity is restored when the split proteins are added back to the protein-deficient cores. Here we show that, among the split proteins, S9 is by far the most effective in restoring S1 binding capability to 3 m-NH₄Cl cores.     

Role of spermidine in the activity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli

The integral membrane protein, sn-glycerol-3-phosphate acyltransferase, catalyzes the first committed step in phospholipid synthesis, and both acyl-CoA and acyl-acyl carrier protein can be used as acyl donors in this reaction. We found that spermidine increased the specific activity of the acyltransferase when either substrate was used as the acyl donor. Magnesium, as well as other cations, also increased acyltransferase activity but were not nearly as effective as spermidine. Two roles for spermidine in this reaction were deduced from our data. First, spermidine dramatically lowered the K_m for glycerol 3-phosphate resulting in an overall rate enhancement when either substrate was used as the acyl donor. This effect was attributed to the modification of the acyl-transferase environment due to the binding of spermidine to membrane phospholipids. A second effect of spermidine was evident only when acyl-acyl carrier protein was used as substrate. Using this acyl donor, a pH optimum of 7.5 was found in the absence of spermidine, but in its presence, the pH optimum was shifted to 8.5. Between pH 7.5 and 8.5, palmitoyl-acyl carrier protein undergoes a conformational change to a more expanded, denatured state and its activity in the acyltransferase assay decreases dramatically. Spermidine restored the native conformation of palmitoyl-acyl carrier protein at pH 8.5, thus accounting for the majority of rate enhancement observed at elevated pH.     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

Neutron-scattering studies of the ribosome of Escherichia coli: a provisional map of the locations of proteins S3, S4, S5, S7, S8 and S9 in the 30 S subunit.

Results of neutron-scattering experiments to determine the distances between seven pairs of proteins within the 30 S ribosomal subunit are presented. These results, combined with earlier data (Engelman et al., 1975; Moore et al., 1977) lead to the construction of a three-dimensional map of the positions of the centers of mass of proteins S3, S4, S5, S7, S8 and S9. The properties of this map and its relationship to other information on the structure of the 30 S subunit are discussed.     

On the control of septation in Escherichia coli.

Mutants of E. coli defective in cell septation (ftsA to ftsG, conditional thermosensitive mutants isolated by Ricard and Hirota) were studied with respect to their membrane protein composition, murein hydrolase activities and rates of synthesis of murein and phospholipids. Three classes of mutants have been distinguished: 1) those affected in both murein and phospholipid synthesis; 2) those affected in either murein or phospholipid synthesis and 3) those affected in neither of these parameters. Overall murein hydrolase activities, after activation, is of the same order in all the mutants screened. In addition to soluble products of murein splitting, we have found insoluble products that appear to be in dynamic equilibrium with the murein of the sacculus. Endogenous levels of cyclic adenosine 3',5'-monophosphate measured after blocking septation showed no variation. This suggests that the cyclic nucleotide is not involved in the metabolic control of septation.     

Binding and reactivity at the "glucose" site of galactosyl-beta-galactosidase (Escherichia coli)

A large number of sugars and alcohols were tested to see how well they bound and how readily they reacted at the "glucose" site of the galactosyl form of beta-galactosidase. Two classes of compounds were found to bind well to the galactosyl form of the enzyme. One class contained sugars and alcohols similar in structure to D-glucose in its pyranose ring form, and the other class was composed of relatively hydrophobic sugars and alcohols. On the other hand, several factors seemed to control k4. Large k4 values were found for straight-chain alcohols as compared to the values for the corresponding ring sugars. Also, if the acceptors had hydroxyl groups at the end of the molecule, the reactivity (k4) was greater than if hydroxyl groups were only in the middle of the molecule. In addition, if there was a hydroxyl at an asymmetric carbon next to a terminal hydroxymethyl group, it was necessary that it be in the same orientation as the D configuration of glucose; otherwise, the k4 was low. Overall, the results showed that it is the binding effect, more than the reactivity, which is responsible for the specificity at the "glucose" site. More specifically, these studies showed that the reason glucose is such an ideal molecule for transgalactosylation is that it leaves the galactosyl form of the enzyme very slowly, that is, k-a is relatively small. Thus, glucose remains attached to the galactosyl form of beta-galactosidase for a sufficient time to allow transgalactosylation to occur, while other acceptors, despite being as reactive (or more reactive) in terms of their k4 values, dissociate from the "glucose" site of the galactosyl form of the enzyme very readily and thus are poor acceptors.     

Ribonucleic acid-protein crosslinking in Escherichia coli ribosomal 30S subunits by the use of two new heterobifunctional reagents: 4-azido-2,3,5,6-tetrafluoropyridine and 4-azido-3,5-dichloro-2,6-difluoropyridine

Two new heterobifunctional reagents (4-azido-2,3,5,6-tetrafluoropyridine and 4-azido-3,5-dichloro-2,6-difluoropyridinel were synthesized and used to crosslink RNA to protein in Escherichia coli ribosomal 30S subunits. The maximal yield of crosslinking of the protein moiety was evaluated as 3.5%. Only proteins S4, S7 and S9 were found to be crosslinked to the 16S RNA within the 30S subunits.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).     

Isolation and characterization of protease do from Escherichia coli, a large serine protease containing multiple subunits

A new cytoplasmic proteolytic enzyme in Escherichia coli, named protease Do, has been purified to near homogeneity. The enzyme is an endoprotease that degrades casein, denatured bovine serum albumin, and globin but shows little or no hydrolytic activity against insulin, growth hormone, native bovine serum albumin, or a variety of commonly used peptide substrates. The molecular size of the enzyme was large, and it could be isolated in different preparations in either of two forms. One showed a molecular weight of about 500,000 on gel filtration and a sedimentation coefficient of 15.9 S on sucrose gradient centrifugation. The other appeared to be about 300,000 and sedimented at 12.7 S. No interconversion between the two forms and no other difference in the properties was found. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) shows that both forms contain a major 54,000-dalton band and three additional minor polypeptides with molecular weights of 45,000, 44,000, and 42,000. These minor polypeptides appear to result from autolytic degradation of the major protein as demonstrated by peptide mapping with Staphylococcus aureus V8 protease. Thus, protease Do appears to contain a single subunit of 54,000, and can exist either as a decamer or as a hexamer or pentamer. The enzyme is a serine protease. It is sensitive to diisopropyl fluorophosphate (DFP) but not to metal chelating agents, sulfhydryl blocking groups, certain chloromethyl ketones, or various peptide aldehyde inhibitors. The enzyme covalently binds [3H]DFP, and the labeled subunit was visualized on SDS-polyacrylamide gels by fluorography. When cells growing in rich broth enter stationary phase, the relative concentration of protease Do increases more than twofold.     

Modes of modifier action in E. coli aspartate transcarbamylase

The observed patterns for inhibition by CTP and succinate of equilibrium exchange kinetics with native aspartate transcarbamylase (E. coli) are consistent with an ordered substrate-binding system in which aspartate binds after carbamyl phosphate, and phosphate is released after carbamyl aspartate. ATP selectively stimulates Asp carbamyl-Asp exchange, but not carbamyl phosphate P_i. Initial velocity studies at 5 °, 15 °, and 35 °C were carried out, using modifiers as perturbants of the system. Modifiers alter the Hill n and S_0.5 for aspartate, most markedly at 15 °C but less so at the other temperatures. ATP does increase V under saturating substrate conditions, and substrate inhibition is observed for aspartate. ATP does not make the Hill n = 1 at any temperature. It is proposed that CTP and ATP act by separate mechanisms, not by simply perturbing in opposite directions the equilibrium for aspartate binding. ATP appears to act to increase the rate of aspartate association and dissociation, whereas CTP induces an intramolecular competitive effect in the protein.