Isolation and crystallization of stable domains of the protein L7/L12 from <i>Escherichia coli</i> ribosomes

Liljas, Anders; Eriksson, Stig; Donner, D.; Kurland, C. G.

doi:10.1016/0014-5793(78)80198-7

Cited by 34 publications

(16 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Liljas et aL (25) have obtained crystals of N-terminal and C-terminal fragments containing amino acids 1-36 and 48-118, respectively. The authors suggest that the spontaneous degradation of L7/L12 in the regions of residues [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] indicates that this region is flexible and accessible to proteolysis.…”

Section: Crystallizationmentioning

confidence: 99%

Chemistry and biology ofE. coli ribosomal protein L12

Brot

Weissbach

1981

Mol Cell Biochem

View full text Add to dashboard Cite

E. coli ribosomal protein L12, because of its unique features, has been studied in more detail than perhaps any of the other ribosomal proteins. Unlike the other ribosomal proteins that are generally present in stoichiometric amounts, there are four copies of L12 per ribosome, some of which are acetylated on the N-terminal serine. The acetylated species, referred to as L7, has not been shown, as yet, to possess any different biological activity than L12. A specific enzyme that acetylates L12 to form L7, using acetyl-CoA as the acetyl donor, has been purified from E. coli extracts. L12 is also unique in that it does not contain cysteine, tryptophan, histidine, or tyrosine, is very acidic (pI: 4.85) and has a high content of ordered secondary structure (approximately 50%). The protein is normally found in solution as a dimer and also forms a tight complex with ribosomal protein L10. There are three methionine residues in L12, located in the N-terminal region of the protein, one or more of which are essential for biological activity. Oxidation of the methionines to methionine sulfoxide prevents dimer formation and inactivates the protein. The four copies of L12 are located in the crest region(s) of the 50S ribosomal subunit. There is good evidence that the soluble factors, such as IF-2, EF-Tu, EF-G and RF, interact with L12 on the ribosome during the process of protein synthesis. This interaction is essential for the proper functioning of each of the factors and for GTP hydrolysis associated with the individual partial reactions of protein synthesis. The L12 gene is located on an operon that contains the genes for L10 and beta beta' subunits of RNA polymerase at about 88 min on the bacterial chromosome. DNA-directed in vitro systems have been used to study the unique regulation of the expression of these genes. Autogenous regulation, translational control, and transcription attenuation are regulatory mechanisms that function to control the synthesis of these proteins.

show abstract

Section: Crystallizationmentioning

confidence: 99%

Chemistry and biology ofE. coli ribosomal protein L12

Brot

Weissbach

1981

Mol Cell Biochem

View full text Add to dashboard Cite

show abstract

“…Noteworthy features of BL17 are that tryptophan and cysteine are absent, a lysine-rich region is present in the N-terminal part @OS. [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], and the hydrophobic amino acids are not clustered but are evenly distributed along the protein chain.…”

Section: Characteristics Of the Sequencementioning

confidence: 99%

“…This approach can now be applied to ribosomal proteins. The C-terminal fragment of E. coli ribosomal proteins L7/L12 has been crystallized [6] and analyzed at a resolution of 2.6 A (A. Liljas, persond commu~cation). The first intact ribosomal protein which could be crystallized f7] is protein BL17 isolated from the large subunit of the Bacillus, sfeurothermophilus ribosome.…”

mentioning

confidence: 99%

The primary structure of protein BL17 isolated from the large subunit of the Bacillus stearothermophilus ribosome

1980

View full text Add to dashboard Cite

To understand, at the molecular level, the function of the ribosome which plays a central role in the biosynthesis of proteins, it is essential to know the structure of the particle in great detail. So far the primary structures of almost all of the 53 proteins [I] and the 3 RNA molecules [Z] present in the Escherichiu coli ribosome are known. Progress has been made towards the elucidation of secondary structures of both the proteins [l] and the RNAs [3-51 also.The most direct way of determining the tertiary structure of a protein is by its crystallization followed by X-ray analysis. This approach can now be applied to ribosomal proteins. The C-terminal fragment of E. coli ribosomal proteins L7/L12 has been crystallized [6] and analyzed at a resolution of 2.6 A (A. Liljas, persond commu~cation). The first intact ribosomal protein which could be crystallized f7] is protein BL17 isolated from the large subunit of the Bacillus, sfeurothermophilus ribosome. The determination of the tertiary structure of this protein by X-ray analysis is now in progress (0. Epp and R. Reinhardt, unpubllshed) .Here, we describe the complete primary structure of protein BL17 with 147 amino acids and present its secondary structure based on 4 prediction programmes. The ammo acid sequence of BL17 is compared to that of other ribosomal proteins. Materials and methodsProtein BL17 was isolated as in [S] avoiding urea and acetic acid extraction. Enzymic digestion was done as follows: ElsevierfNorth-Holland Biomedical Press(i) Trypsin digestion at pH 8 .l (in 0.2 M N-methylmorpholine acetate buffer at 37OC for 3 h); (ii) ArmiZZaria mellea protease digestion at pH 8.1(same buffer as above at 37°C for 6 h); (iii) Pepsin digestion at pH 2 (in 5% formic acid at 37'C for 6 h); (iv) S~a~hyZococcus aureus protease digestion at pH 4.0 (in 0.1 M ammonium acetate buffer at 37°C for 48 h). The resulting peptides were isolated by: (i) One-dimensional preparative thin-layer chromatography (A. metlea protease peptides); (ii) Thin-layer fingerprints (tryptic peptides); (iii) Gel-filtration on Sephadex GSO, superfine(1 X 140 cm) in 10% acetic acid followed by fmgerprints on thin-layer plates (peptic and Staph. aureus peptides). Sequencing of the various peptides was done with the DABITC~~C double-coupling method 19 3. DABITH-Ile and -Leu were determined by onedimensional thin-layer chromatography on polyamide sheets [lo]. Amino acid analysis was done on Durrum D-500 analysersasin [ll]. Results and discussion .l . Sequence determinationTwenty-four peptides were isolated from the tryptic digestion of BLI 7 by thin-layer frn~~~nt~g, and the complete sequences of these peptides (except T7, TlO and T15) were determined. The sequences of the peptides T7 (pas. 32-44), and the order of the tryptic pep tides were obtained from the sequences of the peptic peptides. Sixteen peptides (PI-P16) were isolated 323

show abstract

“…Despite many attempts at crystallization of intact ribosomal protein crystals suitable for X-ray analysis have not been obtained, although the crystallization of two fragments of proteins L7/12 has been reported [4].…”

Section: Introductionmentioning

confidence: 99%