The contacts of yeast tRNA<sup>Ser</sup> with seryl‐tRNA synthetase studied by footprinting experiments

Dock-Brégeon, Anne-Catherine; García, Ángela Domínguez; Giegé, Richard; Moras, Dino

doi:10.1111/j.1432-1033.1990.tb15401.x

Cited by 47 publications

(37 citation statements)

References 57 publications

(19 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The lysate was clarified by centrifugation at 100,000 ϫ g for 90 min at 4°C, giving about 10 ml extract with 10 mg of protein/ml. SerRS was purified by chromatography on DEAE-cellulose (DE52, Whatman) and phosphocellulose (P-11 Whatman) columns, as described previously (20,21). Protein extract was dialyzed against 20 mM potassium phosphate buffer, pH 7.2, containing 10% glycerol, 3 mM DTT, 0.1 mM EDTA, and 0.2 mM PMSF and applied onto a DEAEcellulose column (1.5 ϫ 14 cm) equilibrated in the same buffer.…”

Section: General Procedures-[mentioning

confidence: 99%

“…Full-length SerRS was purified to apparent homogeneity (Fig. 3A, lanes e and f) by a quick two-step chromatographic procedure that involves fractionation on DEAE-cellulose and phosphocellulose columns (20,21) as the important steps in isolation of SerRS from non-overproducing yeast strains. The procedure allows the purification of 1 mg of protein from 1 g of yeast cells, with a specific activity of 115 nmol mg Ϫ1 min…”

Section: Overexpression and Purification Of Wild-type And Truncatedmentioning

confidence: 99%

See 1 more Smart Citation

The C-terminal Extension of Yeast Seryl-tRNA Synthetase Affects Stability of the Enzyme and Its Substrate Affinity

Weygand-Đurašević

Lenhard

Filipić

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) contains a 20-amino acid C-terminal extension, which is not found in prokaryotic SerRS enzymes. A truncated yeast SES1 gene, lacking the 60 base pairs that encode this C-terminal domain, is able to complement a yeast SES1 null allele strain; thus, the C-terminal extension in SerRS is dispensable for the viability of the cell. However, the removal of the C-terminal peptide affects both stability of the enzyme and its affinity for the substrates. The truncation mutant binds tRNA with 3.6-fold higher affinity, while the K m for serine is 4-fold increased relative to the wild-type SerRS. This indicates the importance of the C-terminal extension in maintaining the overall structure of SerRS.Aminoacyl-tRNA synthetases are essential enzymes that catalyze the esterification of an amino acid to its cognate tRNA with exquisite specificity. The combination of biophysical, biochemical and genetic techniques have significantly deepened our understanding of the structure and function of these enzymes (1-3). The amino acid sequences of seryl-tRNA synthetases from Escherichia coli (4), Thermus thermophilus (5), Bacillus subtilis (6), Coxiella burnetii (7), Saccharomyces cerevisiae (8), Chinese hamster (partial) (9), and human 1 are known. According to common structural motifs they are typical representatives of class II synthetases (10). The crystal structures of two prokaryotic enzymes isolated from E. coli (11) and T. thermophilus (12) are quite similar, as is their mode of interaction with tRNA Ser . We have been working with yeast SerRS, 2 which shows only moderate similarity (about 30%) with prokaryotic seryl-tRNA synthetases on the level of the primary structure (5), but still recognizes bacterial tRNA Ser and can functionally substitute for the bacterial enzyme in vivo (13). The overexpression of the yeast SES1 gene in E. coli generated high amounts of functional enzyme (13), although with somewhat lower specific activity and slightly different electrophoretic mobility 3 compared to the enzyme isolated from yeast. This raises the possibility that the yeast enzyme may not be modified or folded correctly in the bacterial host. In this paper we describe the purification of yeast SerRS from an overproducing strain of S. cerevisiae. The alignment of the primary sequences of all SerRS proteins reveals that the enzymes from yeast (8), Chinese hamster (9), and human 1 contain C-terminal extension between 20 and 48 amino acids long not found in prokaryotic synthetases. We speculated whether this peptide was important for maintaining the structure of the eukaryotic enzymes or if it had another function. Thus, we deleted the part of the S. cerevisiae SES1 gene encoding the short C-terminal domain and analyzed the expressed truncated protein. (14). Selection for yeast auxotrophic markers was done in a medium of 0.67% nitrogen base and 2% glucose lacking amino acids, supplemented as needed with adenine (20 g/ml), uracil (20 g/ml), and amino acids (20 -30 g/ml). Sporulation mediu...

show abstract

Section: General Procedures-[mentioning

confidence: 99%

Section: Overexpression and Purification Of Wild-type And Truncatedmentioning

confidence: 99%

The C-terminal Extension of Yeast Seryl-tRNA Synthetase Affects Stability of the Enzyme and Its Substrate Affinity

Weygand-Đurašević

Lenhard

Filipić

et al. 1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The long variable arm has been implicated in the recognition of E. coli tRNA Tyr (Himeno et al 1990) and is an idiosyncratic feature that could interact with the idiosyncratic amino-terminal domain of the CYT-18 protein and contribute to the Neurospora mt tRNA Tyr being a better inhibitor of splicing than is E. coli tRNA Tyr. In the case of yeast tRNA set, which also has a long variable arm, footprinting experiments have shown that SerRS binds to both the variable arm and the base of the anticodon stem (Dock-Bregeon et al 1990). If the situation is similar for the Neurospora mt TyrRS, a key structure involved in recognition may be the cognate of the variable arm, P7, hinged off the cognate of the anticodon arm, P4/P6 (cf.…”

Section: P8mentioning

confidence: 99%

A tyrosyl-tRNA synthetase binds specifically to the group I intron catalytic core.

Guo¹,

Lambowitz

1992

Genes Dev.

View full text Add to dashboard Cite

The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns in mitochondria. Here, we show that CYT-18 binds strongly to diverse group I introns that have minimal sequence homology and recognizes highly conserved structural features of the catalytic core of these introns. Inhibition experiments indicate that the intron RNA and tRNA TM compete for the same or overlapping binding sites in the CYT-18 protein. Considered together with functional analysis, our results indicate that the CYT-18 protein promotes splicing by binding to the intron core and stabilizing it in a conformation required for catalytic activity. Furthermore, the specific binding of the synthetase suggests that the group I intron catalytic core has structural similarities to tRNAs, which could reflect either convergent evolution or an evolutionary relationship between group I introns and tRNAs.[Key Words: Catalytic RNA; group I intron; RNA splicing; aminoacyl-tRNA synthetase; mitochondrial RNA processing]Received April 14, 1992; revised version accepted June 1, 1992.Although group I introns use RNA-catalyzed splicing reactions, many, if not all, require proteins for efficient splicing in vivo (for review, see Lambowitz and Perlman 1990). In the case of mitochondrial RNA splicing, these proteins have been shown to include maturases encoded within some group I introns, as well as a variety of splicing factors encoded by host chromosomal genes. The latter tend to differ between Neurospora and yeast, and they include aminoacyl-tRNA synthetases and other cellular RNA-binding proteins that have an additional function in their host cell. We suggested that host proteins that have already differentiated may have adapted to function in splicing relatively recently in evolution by taking advantage of their ability to recognize structural features of group I introns resembling those in their normal cellular RNA targets (Lambowitz and Perlman 1990). Most host-encoded splicing factors function in splicing one or a small number of related group I introns. However, the Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), functions in splicing a number of different group I introns in mitochondria and presumably recognizes conserved structural features of these introns (Collins and Lambowitz 1985). The fact that the CYT-18 protein is an aminoacyl-tRNA synthetase raises the possibility that 1Corresponding author. these structural features resemble those found in tRNAs (Akins and Lambowitz 1987).To investigate how the Neurospora mt TyrRS functions in splicing, we developed an in vitro-splicing system based on the ability of the protein to splice the Neurospora mitochondrial large rRNA (mt large rRNA) intron, which is not self-splicing under any conditions examined (Garriga and Lambowitz 1986). Studies using this in vitro system showed that splicing occurs by the same guanosine-initiated transesterification reactions used by self-splicing group I introns and remains dependent on the integr...

show abstract

“…Ser have revealed that it contains the major identity element of tRNA Ser (11)(12)(13)(14). The recognition mechanism of SerRS thus appears to be evolutionarily conserved in both prokaryote and eukaryotic cytoplasm.…”

mentioning

confidence: 99%

Dual Mode Recognition of Two Isoacceptor tRNAs by Mammalian Mitochondrial Seryl-tRNA Synthetase

Shimada¹,

Suzuki²,

Watanabe³

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Animal mitochondrial translation systems contain two serine tRNAs, corresponding to the codons AGY (Y ‫؍‬ U and C) and UCN (N ‫؍‬ U, C, A, and G), each possessing an unusual secondary structure; tRNA GCU Ser (for AGY) lacks the entire D arm, whereas tRNA UGA Ser (for UCN) has an unusual cloverleaf configuration. We previously demonstrated that a single bovine mitochondrial seryltRNA synthetase (mt SerRS) recognizes these topologically distinct isoacceptors having no common sequence or structure. Recombinant mt SerRS clearly footprinted at the T⌿C loop of each isoacceptor, and kinetic studies revealed that mt SerRS specifically recognized the T⌿C loop sequence in each isoacceptor. However, in the case of tRNA UGA Ser , T⌿C loop-D loop interaction was further required for recognition, suggesting that mt SerRS recognizes the two substrates by distinct mechanisms. mt SerRS could slightly but significantly misacylate mitochondrial tRNA Gln , which has the same T⌿C loop sequence as tRNA UGA Ser , implying that the fidelity of mitochondrial translation is maintained by kinetic discrimination of tRNAs in the network of aminoacyltRNA synthetases.

show abstract

The contacts of yeast tRNA^Ser with seryl‐tRNA synthetase studied by footprinting experiments

Cited by 47 publications

References 57 publications

The C-terminal Extension of Yeast Seryl-tRNA Synthetase Affects Stability of the Enzyme and Its Substrate Affinity

The C-terminal Extension of Yeast Seryl-tRNA Synthetase Affects Stability of the Enzyme and Its Substrate Affinity

A tyrosyl-tRNA synthetase binds specifically to the group I intron catalytic core.

Dual Mode Recognition of Two Isoacceptor tRNAs by Mammalian Mitochondrial Seryl-tRNA Synthetase

Contact Info

Product

Resources

About

The contacts of yeast tRNASer with seryl‐tRNA synthetase studied by footprinting experiments

Cited by 47 publications

References 57 publications

The C-terminal Extension of Yeast Seryl-tRNA Synthetase Affects Stability of the Enzyme and Its Substrate Affinity

The C-terminal Extension of Yeast Seryl-tRNA Synthetase Affects Stability of the Enzyme and Its Substrate Affinity

A tyrosyl-tRNA synthetase binds specifically to the group I intron catalytic core.

Dual Mode Recognition of Two Isoacceptor tRNAs by Mammalian Mitochondrial Seryl-tRNA Synthetase

Contact Info

Product

Resources

About

The contacts of yeast tRNA^Ser with seryl‐tRNA synthetase studied by footprinting experiments