Release of 70 S ribosomes from polysomes in Escherichia coli

In order to determine whether ribosomes are released from messenger RNA as intact particles or as subunits, polysomes of Escherichia coli labeled with heavy isotopes were allowed to run off together with “light” polysomes. The normally rapid post-run-off exchange of subunits by free ribosomes was virtually eliminated by two means: the use of purified polysomes (relatively free of initiation factors), and incubation at a lower temperature (25 °C), or at a somewhat higher Mg²⁺ concentration (12 to 14 mm), than is conventional. Under these conditions ribosomes released by run-off or by puromycin accumulated without subunit exchange. Hence, even though the ribosome normally initiates via subunits, it is released from RNA by a conformational change in the intact 70 S particle, rather than by dissociation.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

Selective spin-labeling of the ribosomal proteins of 70S ribosomes from Escherichia coli.

We have used a series of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) maleimide spin labels of different length to label, covalently and selectively, the most reactive sulfhydryl groups of 70S ribosomal proteins of Escherichia coli. Under short periods of labeling (1--2 min), less than two spin labels per ribosome are incorporated and were shown to be distributed mainly on five ribosomal proteins in the following order: S18 greater than S21, L27 greater than S17, and S12. With a long period of labeling (3 h) up to 13 spin labels are attached to the ribosome, and protein S1 is the most labeled. The shape of the electron paramagnetic resonance (epr) signal shows two components with a predominance for the strongly immobilized orientation, and the percentage of these components in each spectra has been evaluated. When the distance between the nitroxide group and the maleimide-attaching group exceeds 6 A (1 A = 0.1 nm) the strongly immobilized orientation disappears. The effect of magnesium ions on these selectively spinlabeled ribosomes shows that the dissociation into subunits does not affect the epr signal, but more spin labels are incorporated into the subunits if labeling is performed under conditions of dissociation.     

Identification of neighbouring proteins by cross-linking of intact 70 S ribosomes from Escherichia coli

70 S ribosomes from Escherichia coli have been reacted with the bifunctional reagent 1,4-phenyldiglyoxal under near physiological conditions. As a result of the cross-linking reaction a number of high-molecular-weight protein fractions with altered electrophoretic mobility could be isolated. A new chemical procedure has been introduced to reverse the cross-links between proteins at least partially. The cleavage reaction did not affect the gel electrophoretic mobility of the proteins. Thus a direct identification of cross-linked proteins using one- or two-dimensional gels was made possible. Two protein trimers, S3-S4-S5 and L1-S4-S5, as well as five protein dimers, S3-S4, L6-L7/12, L10-L7/12, S9-L19 and L18-L19 could be identified as close neighbours in the E. coli 70 S ribosome. The protein pairs S9-L19 and L18-L19 had previously not been identified as near neighbours using cross-linking studies.     

Chemical crosslinking of elongation factor G to the 23S RNA in 70S ribosomes from Escherichia coli

Elongation factor G was crosslinked to the 23S RNA of 70S Escherichia coli ribosomes with the bifunctional, cleavable reagent diepoxybutane (DEB). The EF-G-23S RNA complex was isolated and digested with ribonuclease A. After digestion, an RNA fragment, protected by EF-G was cleaved from the complex and isolated. The nucleotide sequence of this RNA fragment was determined by partial ribonuclease digestion. It proved to be 27 nucleotides long and it could be identified with residues 1055 to 1081 of the nucleotide sequence of E. coli 23S RNA. In the presence of thiostrepton, which prevents binding of EF-G to the ribosome, there was a dramatic decrease in the yield of this complex.     

Association of messenger ribonucleic acid with 70S monosomes from down-shifted Escherichia coli.

The complexed 70S ribosomes (monosomes) that accumulate in Escherichia coli after an energy source shift-down were examined in an electron microscope. In all cases, the ribosomes lie at or near one end of a ribonucleic acid (RNA) strand. This messenger RNA (mRNA) has a mean length of 168 nm and a length-average length of 200 nm, sufficient to code for polypeptides of a weight-average molecular weight of 20,000. The length distribution indicates that these strands are a reasonable representation of the population of monocistronic mRNA's of E. coli. The mRNA strands disappear entirely upon digestion with pancreatic ribonuclease, phosphodiesterase I, or polynucleotide phosphorylase. The susceptibility to digestion by 3'-exonucleases indicate that the ribosomes lie at the 5' end of the mRNA strands. These results are consistent with the hypothesis that down-shifted cells have a translational defect at a point subsequent to the binding of ribosomes to mRNA but prior to the formation of the first peptide bond, such that ribosomes remain bound at or near their points of initial attachment to mRNA.     

Analysis of kethoxal bound to ribosomal proteins from Escherichia coli 70S reacted ribosomes

To investigate ribosome topography and possible function, 70S ribosomes of $\text{Escherichia coli}$ were reacted with the dicarbonyl compound kethoxal. Ribosomal protein was extracted after reaction, and through two dimensional gel electrophoresis, the reactive proteins of the two subunits were identified. From the 30S subunit, the most reacted proteins were S2, S3, S4, S5 and S7 and from the 50S subunit, L1, L5, L16, L17, L18 and L27. The results with kethoxal are compared with other modifiers of ribosomal proteins.     

A map of protein-rRNA distribution in the 70 S Escherichia coli ribosome

Neutron scattering exploits the enormous scattering difference between protons and deuterons. A set of 42 x-ray and neutron solution scattering curves from hybrid Escherichia coli ribosomes was obtained, where the proteins and rRNA moieties in the subunits were either protonated or deuterated in all possible combinations. This extensive data set is analyzed using a novel method. The volume defined by the cryoelectron microscopic model of Frank and co-workers (Frank, J., Zhu, J., Penczek, P., Li, Y. H., Srivastava, S., Verschoor, A., Radermacher, M., Grassucci, R., Lata, R. K., and Agrawal, R. K. (1995) Nature 376, 441-444) is divided into 7890 densely packed spheres of radius 0.5 nm. Simulated annealing is employed to assign each sphere to solvent, protein, or rRNA moieties to simultaneously fit all scattering curves. Twelve independent reconstructions starting from random approximations yielded reproducible results. The resulting model at a resolution of 3 nm represents the volumes occupied by rRNA and protein moieties at 95% probability threshold and displays 15 and 20 protein subvolumes in the 30 S and 50 S, respectively, connected by rRNA. 17 proteins with known atomic structure can be tentatively positioned into the protein subvolumes within the ribosome in agreement with the results from other methods. The protein-rRNA map enlarges the basis for the models of the rRNA folding and can further help to localize proteins in high-resolution crystallographic density maps.     

Interaction of N-acetyl-phenylalanyl-tRNAPhe with 70S ribosomes of Escherichia coli.

The interaction of N--Acetyl--Phe--tRNA Phe with 70 S ribosomes is a reversible process in the absence as well as in the presence of messenger. The equilibrium binding constants of these interactions were measured at different magnesium concentrations and temperatures and thermodynamical quantities computed. The enthalpy of the formation of complexes with the P site of ribosomes is larger by 6,000 cal/mol in the presence of poly (U) than in the presence of poly (C) or in total absence of messenger. Free energy differences are rather small, the association constants differ less than one order of magnitude. The association constant of N--Acetyl--Phe--tRNA Phe with the A site of ribosomes is 30--50 times lower than with the P site even in the presence of poly (U).     

Structural study of translating 70 S ribosomes from Escherichia coli: I. Electron microscopy

Translating 70 S ribosomes of Escherichia coli either in the pre-translocation or in the post-translocation state have been prepared by using the cell-free translation system in poly(U)—S—S—Sepharose columns [Methods Enzymol. (1979) 59, 382–398]. Electron microscopy study of the preparations has demonstrated that: (1) the mutual orientation of the ribosomal subunits in the translating ribosomes is the same as proposed by Lake for routine 30 S·50 S couples [J. Mol. Biol. (1976) 105, 111–130]; (2) the L7/L12 stalk of the 50 S subunit sticks out from the 70 S particle and does not join the 30 S subunit; (3) pre-translocation and post-translocation state ribosomes do not differ in mutual orientation of the subunits and in the position of the L7/L12 stalk, within the limits of electron microscopy resolution.     

Composition of the Escherichia coli 70S ribosomal interface: a cross-linking study

70S tight-couple ribosomes from Escherichia coli were cross-linked by using the bifunctional reagent phenyl-diglyoxal (PDG). The reaction was stopped after 4-h incubation while still in the linear range. In comparison with untreated ribosomes, 30% of those treated with PDG were shown, by sucrose gradient experiments, not to be separable into their subunits, but remained as 70S particles. There was no detectable change in the structure of the reacted particles when their sedimentation behavior was compared with that of native 70S controls. When the cross-linking reaction was performed in the presence of tRNAPhe and poly(U), the reacted ribosomes retained 40-50% of their tRNA binding activity. The reaction leads predominantly to the formation of RNA-protein cross-links but protein--protein as well as RNA-RNA cross-links could also be detected. Cross-linked material was extracted, and the individual RNAs were separated into 23S, 16S, and 5S RNAs. Proteins were identified electrophoretically after reversal of the RNA-protein cross-links. Proteins were found to be cross-linked to RNAs within and across the ribosomal subunits; the latter are considered to be close to or at the 70S subunit interface. The arrangement of RNA and protein at the subunit interface is discussed.     

Zn(II) binding to Escherichia coli 70S ribosomes

Escherichia coli 70S ribosomes tightly bind 8 equiv of Zn(II), and EXAFS spectra indicate that Zn(II) may be protein-bound. Ribosomes were incubated with EDTA and Zn(II), and after dialysis, the resulting ribosomes bound 5 and 11 equiv of Zn(II), respectively. EXAFS studies show that the additional Zn(II) in the zinc-supplemented ribosomes binds in part to the phosphate backbone of the ribosome. Lastly, in vitro translation studies demonstrate that EDTA-treated ribosomes do not synthesize an active Zn(II)-bound metalloenzyme, while the as-isolated ribosomes do. These studies demonstrate that the majority of intracellular Zn(II) resides in the ribosome.     

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.     

Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli

The RNA degradosome is a multi-enzyme assembly that contributes to key processes of RNA metabolism, and it engages numerous partners in serving its varied functional roles. Small domains within the assembly recognize collectively a diverse range of macromolecules, including the core protein components, the cytoplasmic lipid membrane, mRNAs, non-coding regulatory RNAs and precursors of structured RNAs. We present evidence that the degradosome can form a stable complex with the 70S ribosome and polysomes, and we demonstrate the proximity in vivo of ribosomal proteins and the scaffold of the degradosome, RNase E. The principal interactions are mapped to two, independent, RNA-binding domains from RNase E. RhlB, the RNA helicase component of the degradosome, also contributes to ribosome binding, and this is favoured through an activating interaction with RNase E. The catalytic activity of RNase E for processing 9S RNA (the ribosomal 5S RNA precursor) is repressed in the presence of the ribosome, whereas there is little affect on the cleavage of single-stranded substrates mediated by non-coding RNA, suggestings that the enzyme retains capacity to cleave unstructured substrates when associated with the ribosome. We propose that polysomes may act as antennae that enhance the rates of capture of the limited number of degradosomes, so that they become recruited to sites of active translation to act on mRNAs as they become exposed or tagged for degradation.