Separation of ribosomal proteins from Escherichia coli and rabbit reticulocytes using reverse-phase high-performance liquid chromatography

Ferris, Robert J.; Cowgill, Cynthia; Traut, Robert R.

doi:10.1021/bi00310a009

Cited by 30 publications

(4 citation statements)

References 23 publications

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“…By adapting the approach used by Ferris et al (1984), we separated EXT into 26 distinct peaks ( Fig. 4A) containing at least 36 proteins (data not shown).…”

Section: Ext Consists Of Multiple Proteinsmentioning

confidence: 99%

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Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.

Coetzee¹,

Herschlag²,

Belfort³

1994

Genes Dev.

154

143

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To address the effect of host proteins on the self-splicing properties of the group I introns of bacteriophage T4, we have purified an activity from EscherJchia coil extracts that facilitates both trans-and c/s-splicing of the T4 introns in vitro. The activity is attributable to a number of proteins, several of which are ribosomal proteins. Although these proteins have variable abilities to stimulate splicing, ribosomal protein S12 is the most effective. The activity mitigates the negative effects on splicing of the large internal open reading frames (ORFs) common to the T4 introns. In contrast to proteins shown previously to facilitate group I splicing, S12 does not bind strongly or specifically to the intron. Rather, S12 binds RNA with broad specificity and can also facilitate the action of a hammerhead ribozyme. Addition of S12 to unreactive trans-splicing precursors promoted splicing, suggesting that S12 can resolve misfolded RNAs. Furthermore, incubation with $12 followed by its proteolytic removal prior to the initiation of the splicing reaction still resulted in splicing enhancement. These results suggest that this protein facilitates splicing by acting as an RNA chaperone, promoting the assembly of the catalytically active tertiary structure of ribozymes. [ Bacteriophage T4 contains three self-splicing group I in-trons located in the structural genes for thymidylate syn-thase (td), ribonucleotide reductase (nrdB), and a putative anaerobic ribonucleotide reductase (sunY} (Chu et al. 1984; Gott et al. 1986; Shub et al. 1987; Sun et al. 1993). These introns splice by the typical group I pathway, via two transesterification reactions initiated by nucleo-philic attack of guanosine at the 5' splice site. This process depends on conserved secondary and tertiary structures that direct folding of the intron such that the 5' and 3' splice sites are juxtaposed to the guanosine-binding site within the intron's catalytic core (Cech 1990; Michel and Westhof 1990; Cech et al. 1992; Saldanha et al. 1993). Although a number of group I introns (including the T4 introns} self-splice in vitro, evidence points to involvement of accessory factors during in vivo splicing. 4present address: Brain and

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“…By adapting the approach used by Ferris et al (1984), we separated EXT into 26 distinct peaks ( Fig. 4A) containing at least 36 proteins (data not shown).…”

Section: Ext Consists Of Multiple Proteinsmentioning

confidence: 99%

“…EXT proteins were purified by reverse-phase HPLC using methods described by Cooperman et al (1988) and Ferris et al (1984). HPLC was performed on a system consisting of two Isco 2350 pumps and a V 4 UV detector, which was controlled by a PC computer running Chemsearch Software (Isco).…”

Section: Purification Of Ext Proteinsmentioning

confidence: 99%

Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.

Coetzee¹,

Herschlag²,

Belfort³

1994

Genes Dev.

154

143

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“…Preparation of ribosomes and ribosomal proteins was performed as described previously (Milne et al, 1975). The extracted proteins in 66 % (v/v) acetic acid containing 30 mM-magnesium acetate were injected directly into a Synchropak RP-P C18 column as suggested by Ferris et al (1984). H.p.l.c.…”

Section: Ribosomal Proteinsmentioning

confidence: 99%

Suppression of fluorescence of tryptophan residues in proteins by replacement with 4-fluorotryptophan

Bronskill

Wong

1988

Biochemical Journal

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The tryptophan-auxotrophic Bacillus subtilis LC33 mutant strain utilizes either tryptophan or 4-fluorotryptophan for growth. Proteins therefore could be isolated from these cells in either tryptophan-containing or 4-fluorotryptophan-containing forms. Since 4-fluorotryptophan is non-fluorescent, tryptophan fluorescence would be suppressed in the 4-fluorotryptophan-containing proteins, facilitating the investigation of other chromophores either on the proteins or interacting with the proteins. This approach, potentially applicable to any protein endogenous to or clonable into B. subtilis, was illustrated by an examination of the fluorescence of B. subtilis ribosomal proteins.

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“…Reverse-phase h.p.l.c., on large-pore C,8-silicacolumns, has been used successfully to separate mixtures of peptides, polypeptides and proteins, and in particular has provided a rapid, sensitive and highly reproducible method for resolution of r-proteins from Escherichia coli (Kerlavage et al, 1983), rabbit reticulocytes (Ferris et al, 1984) and yeast (Kerlavage et al, 1985). Kerlavage et al (1983) considered the possibility of using ion-exchange h.p.l.c.…”

Section: Resultsmentioning

confidence: 99%

Application of high-performance liquid chromatography to the purification and characterization of ribosomal protein L3 from trichodermin-resistant yeast mutants

Threadgill¹,

Conrad²,

Changchien³

et al. 1986

Biochemical Journal

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A new h.p.l.c. cation-exchange method has been used to separate proteins from 60S ribosomal subunits prepared from strains of Saccharomyces cerevisiae sensitive or resistant to trichodermin. Ribosomal protein L3 was identified in column eluates by one-dimensional and two-dimensional gel electrophoresis and purified further by reverse-phase h.p.l.c. The protein was cleaved with CNBr and the products were analysed, again by reverse-phase h.p.l.c. A marked difference was observed in the peptide profiles between preparations from trichodermin-sensitive and trichodermin-resistant yeast strains. These results provide the first direct demonstration that, in yeast, mutationally induced resistance to trichodermin can alter the covalent structure of ribosomal protein L3. They convincingly demonstrate the potential of the experimental technique for the rapid and preparative separation of a selected yeast ribosomal protein and its subsequent characterization.

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Separation of ribosomal proteins from Escherichia coli and rabbit reticulocytes using reverse-phase high-performance liquid chromatography

Cited by 30 publications

References 23 publications

Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.

Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.

Suppression of fluorescence of tryptophan residues in proteins by replacement with 4-fluorotryptophan

Application of high-performance liquid chromatography to the purification and characterization of ribosomal protein L3 from trichodermin-resistant yeast mutants

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