The primary structure of protein L10 from <i>Escherichia coli</i> ribosomes

Dovgas, N.V.; Vinokurov, Leonid M.; Velmoga, I.S.; Alakhov, Yu.B.; Ovchinnikov, Yu.A.

doi:10.1016/0014-5793(76)80870-8

Cited by 16 publications

(4 citation statements)

References 7 publications

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“…For this reason, we kept L10 throughout the preparation in 6 M urea until the final experiments, e.g., reconstitution (see Experimental Procedures). L10 has one cysteine at position 70 (Heiland et al, 1976;Dovgas et al, 1976). In accordance with the fact that maleimides will react at pH 6.0 primarily with sulfhydry!…”

Section: Resultsmentioning

confidence: 90%

Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli

Zantema¹,

Maassen²,

Kriek³

et al. 1982

Biochemistry

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So that the topographic and dynamic properties of the L7/L12--L10 complex in the 50S ribosome of Escherichia coli could be studied, methods and reagents were developed in order to introduce fluorescent groups at specific positions of these proteins. In the case of L7/L12, this was done by attaching an aldehyde group to Lys-51 of the protein by using 4-(4-formylphenoxy)butyrimidate or by converting the amino terminus of L12 into an aldehyde group by periodate oxidation. Subsequent reaction of the aldehyde groups with newly developed hydrazine derivatives of fluorescein and coumarin resulted in specifically labeled L7/L12 derivatives. L10 was modified at the single cysteine residue with N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide. The fluorescent proteins L10 and L7/L12 could be reconstituted into 50S ribosomes. The resulting specifically labeled 50S ribosomes show 25--100% activity in elongation factor G dependent GTPase as well as in polyphenylalanine synthesis. The fluorescent properties of the labeled 50S ribosomes show that these fluorescent derivatives are suitable for energy transfer studies.

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Section: Resultsmentioning

confidence: 90%

Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli

Zantema¹,

Maassen²,

Kriek³

et al. 1982

Biochemistry

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“…The tritiated proteins served as internal standards for the estimation of the recovery of the synthesized products after immunoprecipitation and gel electrophoresis. The amount of the in vitro products formed was determined from the specific activity of the amino acid used and the amino acid composition of the individual proteins (25)(26)(27)(28)(29).' It has recently been shown that the ascites extract contains a factor that simulates the in vitro synthesis of 13-galactosidase, very likely by protecting mRNA against degradation (16).…”

Section: Uniformlymentioning

confidence: 99%

DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

Zarucki-Schulz¹,

Jerez²,

Goldberg³

et al. 1979

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

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The in vitro synthesis of elongation factor (EF)-Tu (tufB), the #I' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage Xrifdl8, EF-Tu (tufA), EF-G, and the a subunit of RNA polymerase directed by DNA from the transducing phage Xfus3 has been investigateydin a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the a and Y' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates gB-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the ,' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results.DNA-directed in vitro protein-synthesizing systems afford a valuable tool with which to study the regulation of gene expression (for review, see ref. 1). Because of the complexity of the system, these in vitro studies have used relatively crude extracts to obtain protein synthesis. However, a great deal of new information might be obtained if gene expression could be achieved in a more defined system. Our laboratory has initiated studies on the DNA-directed in vitro synthesis of 13-galactosidase with the hope of obtaining synthesis of this protein in a completely defined system (2-7). It has been possible to obtain significant synthesis of /3-galactosidase in a partially defined system that contained many highly purified factors in addition to several partially purified fractions (7).In conjunction with the studies on the synthesis of 13-galactosidase, experiments were initiated on the expression of the two elongation factor (EF)-Tu genes, tufA and tufB (Fig. 1), carried by the transducing phages Xfus3 and Xrifdl8, respectively (8,9). In addition, quantitative assays have been developed to measure the expression of other genes on these phages, such as ribosomal proteins L10 and L12, EF-G, and the a and 13' subunitst of RNA polymerase (see Fig. 1). The results of these studies using a crude and partially defined system are described here. Bacteriophages Xrifdl8 and Xfus3 were isolated from Escherichia coil H105 (J. B. Kirschbaum, Harvard University) and E. coli N01380 (M. Nomura, University of Wisconsin), respectively. The phages were purified and DNA was extracted as described (10, 11). Preparation, from E. coli Z19iq (provided by G. Zubay, Columbia University), of the ribosomal wash, washed ribosomes, and a supernatant extract (S-200) has been reported (2, 12). The S-200 ...

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“…REAGENT CONCENTRATION L7/L12 molecules (23) and 13 in the L10 molecule (24,25). However, modification of L7/L12 using another arginine-specific reagent, cyclohexanedione, has been reported (26) and it does not influence the dimeric structure of L7/L12 or the ability to bind back to core particles.…”

mentioning

confidence: 99%

Studies on the RNA and protein binding sites of the E.coli ribosomal protein L10

Pettersson

1979

Nucl Acids Res

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We have used modification of specific amino acid residues in the E. coli ribosomal protein L10 as a tool to study its interactions with another ribosomal protein, L7/L12, as well as with ribosomal core particles and with 23S RNA. The ribosome and RNA binding capability of L10 was found to be inhibited by modification of one more of its arginine residues. This treatment does not affect the ability of L10 to bind four molecules of L7/L12 in a L7/L12-L10 complex. Our results support the view that L10's role in promoting the L7/L12-ribosome association is due primarily to its ability to bind to both 23S RNA and L7/L12 simultaneously.

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The primary structure of protein L10 from Escherichia coli ribosomes

Cited by 16 publications

References 7 publications

Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli

Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli

DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

Studies on the RNA and protein binding sites of the E.coli ribosomal protein L10

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