Ribosomal proteins S5, S11, S13 and S19 localized by ekectron microscopy of antibody-labeled subunits

Lake, James A.; Kahan, Lawrence

doi:10.1016/s0022-2836(75)80177-x

Cited by 112 publications

(38 citation statements)

References 41 publications

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“…Sedimentation equilibrium experiments gave molecular weight values that agreed with those obtained from the amino acid sequence [20,21] , namely 12 257 for S8 and 18 839 for L6. Figure 1 shows sedimentation equilibrium experiments with the four proteins.…”

Section: Resultssupporting

confidence: 72%

“…Protein concentrations were determined by three methods: (1) A fluorescamine assay using lysozyme as a standard ]18]-(2) Hydrolysis of aliquots of the protein solution with 6 N hydrochloric acid containing 0.2% 2-mercaptoethanol under nitrogen at 1 10°C for 20 h, followed by amino acid analysis on a Durrum D-500 analyzer. (3) A nitrogen assay [ 191 using ammonium sulfate as a standard and calculation of the nitrogen content of each protein from its amino acid sequence [20,21] . …”

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confidence: 99%

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Hydrodynamic studies on the Escherichia coli ribosomal proteins S8 and L6, prepared by two different methods

1977

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Several studies have been made to examine the shape of individual ribosomal proteins both in situ on the surface of the ribosome and in an isolated state. The immunolo~cal approach to establish the number of specific antibody binding sites for individual proteins on the surface of the ribosome has revealed preliminary evidence for the shape of many protein molecules f 1 ,2] . Hydrodyn~ic studies have previously been performed on proteins S3, S4, S5, S7 and S20 [3,4] and proteins L7/12 [5]. Further information on the shape of these proteins has come from low-angle X-ray diffraction studies with proteins 54 [4,6], L18 and L25 [7] and L7/12 and LlO [8,9] and from neutron scattering experiments with proteins S2, S5 and S8 [lo].In this study proteins S8 and L6 have been investigated by molecular weight determination, sedimentation and diffusion coefficients and viscosity measurements. Both proteins have been prepared by two different methods: one involving the use of urea at low pH and the other using non-denaturing conditions avoiding the use of urea. Protein S8 prepared by both methods was found to be a compact spherical molecule whereas protein L6 appeared to have a slightly elongated shape. A difference in sedimentation was observed between the two L6 preparations showing that the protein isolated under non-denaturing conditions was more compact. Both proteins showed a reversed solubility effect when prepared by the two different methods. 2. Materials and methodsProteins were prepared according to the following two methods: (1) In the presence of urea and acetic acid [ 11,121. (2) In the absence of urea, acetic acid and other potentially denaturing conditions at all steps of the isolation [13,14]. The proteins were extracted from the ribosomal subunits into several groups by a stepwise increase of LiCl at neutral pH. Further purification was achieved by chromatography on CM-Sephadex C-25 and Sephadex G-l 00 in the presence of salt and in the pH-range 5.5-8.0. Purified proteins were concentrated by pressure ultrafiltration or dialysis against dry Sephadex G-l 50. The identity and purity of the proteins was established by two-dimensional gel electrophoresis [ 151 and by dodecylsulphate slab-gel electrophoresis [ 16,17 J . Protein concentrations were determined by three methods: (1) A fluorescamine assay using lysozyme as a standard ]18]-(2) Hydrolysis of aliquots of the protein solution with 6 N hydrochloric acid containing 0.2% 2-mercaptoethanol under nitrogen at 1 10°C for 20 h, followed by amino acid analysis on a Durrum D-500 analyzer. (3) A nitrogen assay [ 191 using ammonium sulfate as a standard and calculation of the nitrogen content of each protein from its amino acid sequence [20,21] .

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Section: Resultssupporting

confidence: 72%

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confidence: 99%

Hydrodynamic studies on the Escherichia coli ribosomal proteins S8 and L6, prepared by two different methods

1977

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“…Those sites functioning in translation are clustered into part of the ribosome, composing approximately two-thirds of its volume, that we have named the "translational domain. " Functional sites contained in the translational domain include: the initiation factor binding sites (1)(2)(3)(4) located in the cleft of the small subunit, the messenger binding sites located on the platform of the small subunit (3,(5)(6)(7), the peptidyltransferase and the 5S RNA located on the central protuberance of the large subunit (8)(9)(10)(11), and proteins mediating the GTP-dependent steps of translation that are found on the L7/L12 stalk of the large subunit (12). Together, these sites define the translational domain.…”

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confidence: 99%

Nascent polypeptide chains emerge from the exit domain of the large ribosomal subunit: immune mapping of the nascent chain.

Bernabéu¹,

Lake²

1982

Proc. Natl. Acad. Sci. U.S.A.

128

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The site of the nascent polypeptide chain as it leaves the ribosome has been localized on the "exit domain" of the Escherichia coli ribosome by using IgG antibodies directed against the enzyme 13-galactosidase (EC 3.2.1.23). Thus, a functional site has been mapped on intact 70S ribosomes. The exit site is on the large subunit, approximately 70 A from the interface between subunits and nearly 150 A from the central protuberance, the likely site of peptide transfer. It is adjacent to the region corresponding to the rough endoplasmic membrane binding region ofthe eukaryotic ribosome but distant from ribosomal components participating in mRNA recognition and polypeptide elongation (ie., distant from the "translational domain"). These results, together with the protease protection experiments of others, provide evidence that the nascent protein chain probably passes through the ribosome in an unfolded, fully extended conformation.The ribosome, as the organelle that translates genetic messages into proteins, has roles both in the translation of the message and in the manufacture and secretion ofthe protein. Currently, a good deal is known about the biochemistry of ribosomal components and factors, but this knowledge is only now being integrated with that of ribosomal three-dimensional structure. Although the locations of many of the ribosomal components involved in the translation ofthe code are known, little is known about the location and path of the protein chain as it passes through the ribosome. Those sites functioning in translation are clustered into part of the ribosome, composing approximately two-thirds of its volume, that we have named the "translational domain. " Functional sites contained in the translational domain include: the initiation factor binding sites (1-4) located in the cleft of the small subunit, the messenger binding sites located on the platform of the small subunit (3,(5)(6)(7), the peptidyltransferase and the 5S RNA located on the central protuberance of the large subunit (8)(9)(10)(11), and proteins mediating the GTP-dependent steps of translation that are found on the L7/L12 stalk of the large subunit (12). Together, these sites define the translational domain.In this paper, we have investigated the location ofthe nascent chain as it emerges from the ribosome, a function associated with protein secretion rather than the translation steps just described. Using antibodies directed against the enzyme ,B-galactosidase (EC 3.2.1.23) to map the exit site ofthe nascent protein chain, we find that it exits from the ribosome at a single region, located on the large subunit. This region is distant from the translational domain and is approximately 150 A from the presumed site of the peptidyltransferase (8,9). No functional site has been mapped directly on 70S ribosomes previously, and in addition the site is found on a region of the 50S subunit where no proteins or functions have been previously found. The corresponding region of the eukaryotic ribosome (13, 14), however, contains a ribos...

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“…Electron microscopy studies of Lake et al [26], using antibody markers, show that S 13 and S 19 are located side by side on the head of the 30 S (3) (4) (5) subunit, although they were found to be more separated in a similar study by Tischendorf et al…”

Section: Discussionmentioning

confidence: 86%

The RNA binding properties of ‘native’ protein—protein complexes isolated from the Escherichia coli ribosome

Dijk¹,

Littlechild²,

Garrett³

1977

FEBS Letters

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Ribosomal proteins S5, S11, S13 and S19 localized by ekectron microscopy of antibody-labeled subunits

Cited by 112 publications

References 41 publications

Hydrodynamic studies on the Escherichia coli ribosomal proteins S8 and L6, prepared by two different methods

Hydrodynamic studies on the Escherichia coli ribosomal proteins S8 and L6, prepared by two different methods

Nascent polypeptide chains emerge from the exit domain of the large ribosomal subunit: immune mapping of the nascent chain.

The RNA binding properties of ‘native’ protein—protein complexes isolated from the Escherichia coli ribosome

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