lysyl-tRNA Synthetase Gene Research Articles

Improving the accuracy of recombinant protein production through integration of bioinformatics, statistical and mass spectrometry methodologies

Heterologous protein production is a key technology for biotechnological, health sciences and many other research fields. Various approaches have been developed for its optimization, but the research emphasis has been on optimization of protein yield rather than protein quality. In this study, we have established a workflow for synthetic gene optimization for heterologous protein expression that combines bioinformatics, laboratory experiments, mass spectrometry and statistical analysis. Two gene primary structure analysis platforms, Anaconda and EuGene, and multivariate optimization methods were employed to re-design the Plasmodium falciparum lysyl-tRNA synthetase gene for optimal expression in Escherichia coli. Synthetic genes were expressed from common vectors, and amino acid mis-incorporations in the expressed proteins were detected and quantified using mass spectrometry. The association between the identified amino acid mis-incorporations and 23 gene variables was then analysed. The synthetic genes yielded significantly higher levels of protein relative to the wild-type gene, but 71 amino acid mis-incorporation sites were observed along the whole protein and across the synthetic genes that were statistically associated with specific codons and protein secondary structures. The optimization method that led to production of the most accurate protein was based on a multivariate approach that combined variables that are known to influence mRNA translation.

The Human Lysyl-tRNA Synthetase Gene Encodes Both the Cytoplasmic and Mitochondrial Enzymes by Means of an Unusual Alternative Splicing of the Primary Transcript

Two cDNAs encoding human lysyl-tRNA synthetase have been identified. One encodes the cytoplasmic form of the enzyme identified previously. The second cDNA contains the same sequence but with a 180-bp insertion at the 5'-end of the mRNA. This results in a predicted protein whose carboxyl 576 amino acids are identical to those of the cytoplasmic enzyme but with a different amino terminus of 49 amino acids that contains a putative mitochondrial targeting sequence. Expression of the two lysyl-tRNA synthetase-green fluorescent protein gene fusions in a human cell line confirmed that the cytoplasmic form was targeted to the cytoplasm and the mitochondrial form to mitochondria. The genomic lysyl-tRNA synthetase gene consisted of 15 exons. The two isoforms were created by alternative splicing of the first three exons of the gene. The cytoplasmic form was created by splicing exon 1 to exon 3. The inclusion of exon 2 between exons 1 and 3 produced an mRNA encoding the mitochondrial isoform with an additional upstream small open reading frame, consisting mainly of a portion of the 5' coding region of the cytoplasmic isoform. This is the first example of mitochondrial targeting sequence being encoded on the second exon of a gene. Ribonuclease protection analysis showed that the mRNA encoding the cytoplasmic isoform makes up approximately 70%, and the mitochondrial isoform approximately 30%, of the mature transcripts from the lysyl-tRNA synthetase gene. The mitochondrial form of the enzyme, purified after expression in Escherichia coli, aminoacylated in vitro transcripts corresponding to both the cytoplasmic and mitochondrial tRNA(Lys), despite the difference in the discriminator base sequence in the acceptor stems of these tRNAs.

The DB case: pattern matching evidence is not significant. MicroCorrespondence

The DB case: pattern matching evidence is not significant. MicroCorrespondence

The trp RNA-binding attenuation protein (TRAP) regulates the steady-state levels of transcripts of the Bacillus subtilis folate operon.

The Bacillus subtilis folate operon contains nine genes. The first six genes are involved in the biosynthesis of folic acid and tryptophan and have been characterized previously. The 3'-region of the folate operon contains three additional ORFs: orf3, potentially encoding a DNA-binding protein of 68 amino acids, orf4, encoding a protein of 338 amino acids with homology to the Orf1 of the E. coli fis operon, and a putative lysyl-tRNA synthetase gene (LysS). Four transcripts were identified which encode the first two, eight or all nine proteins or only the last protein LysS. The folate operon contains two promoters, one upstream of the first gene and the second preceding LysS. Transcription of the entire folate operon starts 33 bp upstream of the ATG codon of pab, the first gene of the operon. The mtrB-encoded trp RNA-binding attenuation protein (TRAP) dramatically reduces the steady-state levels of the folate operon transcripts encoding the first eight and all nine proteins, but only has a relatively small effect on the steady-state level of the 2.1 kb transcript encoding the first two genes of the operon, pab and trpG. In addition, transcription of the folate operon is regulated in a growth-phase-dependent manner. Transcripts were present in very low levels after mid-exponential phase, but were dramatically increased directly after transfer of the cells to fresh medium. These results indicate that transcription of the folate operon is regulated by TRAP and also depends on the growth phase of the culture.

On the nature and use of randomly amplified DNA from Staphylococcus aureus.

Various DNA-based methods have been introduced to genetically type Staphylococcus aureus strains but not a single technique is universally applicable. In order to search for DNA probes suitable for differentiating strains, randomly amplified polymorphic DNA patterns were generated for 243 S. aureus strains and a single isolate of Staphylococcus intermedius. All fingerprints were examined for unique amplicons, and the nature of 42 of these DNA fragments was investigated. Partial DNA sequences were determined, and several homologies were discovered with known S. aureus sequences (plasmid pSH6 DNA with insertion sequences, agrA and agrB sequences, hld genes, the gene for 23S rRNA, the lysyl tRNA synthetase gene, and the threonyl tRNA synthetase gene) and with genes from other species (Haemophilus influenzae bexA and Bacillus subtilis spoF and ctrA). Thirty fragments were of previously unknown origin. In Southern blots containing Eco RI-digested DNA from S. aureus strains and the S. intermedius strain, nine probes demonstrated the capacity to differentiate strains on the basis of the presence or absence of the sequence element in the staphylococcal genome involved. The remainder of the probes displayed restriction fragment length polymorphisms (n = 12), hybridized in a homogeneously positive fashion (n = 13) or hybridized only with their source strains (n = 8) (four of the latter were specific to S. intermedius). Three of the nine strain-specific probes were overlapping, and two of the others were found to display a high level of inconsistency among epidemiologically related strains. Thus, five strain-specific probes remained that, in a 5-digit typing system, accurately distinguished epidemiologically related and unrelated strains of S. aureus. We conclude that application of strain-specific DNA probes, selected on the basis of differing randomly amplified polymorphic DNA patterns, promises to become a technically simple, robust, and reproducible tool that may significantly facilitate the study of the epidemiology of S. aureus infections.

The occurrence of duplicate lysyl-tRNA synthetase gene homologs in Escherichia coli and other procaryotes

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E. coli K-12 and B; E. coli reference collection (ECOR) isolates EC2, EC49, EC65, and EC68; Shigella flexneri; Salmonella typhimurium; Klebsiella pneumoniae; Enterobacter aerogenes; Serratia marcescens; and Proteus vulgaris and the nonenterics Pseudomonas aeruginosa and Bacillus megaterium were grown in AC broth to a pH of 5.5 or less or cultured in SABO medium at pH 5.0. These growth conditions are known to induce LysRS activity (LysU synthesis) in E. coli K-12. Significant induction of LysRS activity (twofold or better) was observed in the E. coli strains, the ECOR isolates, S. flexneri, K. pneumoniae, and E. aerogenes. To demonstrate an association between LysRS induction and two distinct LysRS genes, Southern blotting was performed with a probe representing an 871-bp fragment amplified from an internal portion of the coding region of the lysU gene. In initial experiments, chromosomal DNA from E. coli K-12 strain MC4100 (lysS+ lysU+) was double digested with either BamHI and HindIII or BamHI and SalI, producing hybridizable fragments of 12.4 and 4.2 kb and 6.6 and 5.2 kb, respectively. Subjecting the chromosomal DNA of E. coli K-12 strain GNB10181 (lysS+ delta lysU) to the same regimen established that the larger fragment from each digestion contained the lysU gene. The results of Southern blot analysis of the other bacterial strains revealed that two hybridizable fragments were obtained from all of the E. coli and ECOR collection strains examined and S. flexneri, K. pneumoniae, and E. aerogenes. Only one lysU homolog was found with S. typhimurium and S. marcescens, and none was obtained with P. vulgaris. A single hybridizable band was found with both P. aeruginose and B, megaterium. These results show that the dual-gene LysRS system is not confined to E. coli K-12 and indicate that it may have first appeared in the genus Enterobacter.

Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms

We cloned the GCD5 gene of S. cerevisiae and found it to be identical to KRS1, which encodes lysyl-tRNA synthetase (LysRS). The mutation gcd5-1 changes a conserved residue in the putative lysine-binding domain of LysRS. This leads to a defect in lysine binding and, consequently, to reduced charging of tRNA Lys. Mutant gcd5-1 cells compensate for the defect in LysRS by increasing GCN4 expression at the translational level. GCN4 protein in turn stimulates transcription of GCD5, leading to increased LysRS activity. We propose an autoregulatory model in which uncharged tRNA Lys stimulates the protein kinase GCN2, a translational activator of GCN4, and thereby increases transcription of GCD5 and other genes regulated by GCN4.

Lysyl-tRNA synthetase gene of Campylobacter jejuni

We report the cloning and complete nucleotide sequence of the Campylobacter jejuni lysyl-tRNA synthetase gene (lysS). The C. jejuni lysS gene sequence shows high homology to the two Escherichia coli lysyl-tRNA synthetase genes, lysS and lysU. The Campylobacter lysyl-tRNA synthetase protein (LysRS) shows 47.9 and 46.6% sequence identity to the E. coli enzymes encoded by the lysS and lysU genes, respectively. The LysRS encoded by the C. jejuni gene is a polypeptide of 501 amino acids with a deduced molecular weight of 57,867. The enzyme is active in E. coli. The gene is expressed from its own promoter, and the transcription start site has been mapped. The carboxyl-terminal codon of the C. jejuni lysS gene overlaps by 1 bp with the Met initiation codon of the glyA gene, which has been shown to have a promoter which is functional in E. coli (V.L. Chan and H.L. Bingham, Gene 101:51-58, 1991). C. jejuni, unlike E. coli, has only one lysyl-tRNA synthetase gene.

APMR2 tandem repeat with a modified C-terminus is located downstream from theKRS1 gene encoding lysyl-tRNA synthetase inSaccharomyces cerevisiae

The KRS1 gene encodes the cytoplasmic form of Saccharomyces cerevisiae lysyl-tRNA synthetase. The KRS1 locus has been characterized. The lysyl-tRNA synthetase gene is unique in the yeast genome. The gene is located on the right arm of chromosome IV and disruption of the open reading frame leads to lethality. These results contrast with the situation encountered in Escherichia coli where lysyl-tRNA synthetase is coded by two distinct genes, lysS and lysU, and further address the possible biological significance of this gene duplication. The nucleotide sequence of the 3'-flanking region has been established. It encodes a long open reading frame whose nucleotide and amino acid structures are almost identical to PMR2, a cluster of tandemly repeated genes coding for P-type ion pumps. The sequence alterations relative to PMR2 are mainly located at the C-terminus of the protein.

The yeast lysyl-tRNA synthetase gene. Evidence for general amino acid control of its expression and domain structure of the encoded protein.

The nucleotide sequence of a 3.6-kilobase pair DNA fragment containing the structural gene for yeast cytoplasmic lysyl-tRNA synthetase (KRS1) and its flanking regions was determined. The encoded protein of 67,881 kDa displays a cluster of 11 lysines within a 29-amino acid residue segment at its amino-terminal extremity. Evidence is presented that this segment is responsible for the affinity displayed by the native enzyme toward polyanionic carriers. The transcription initiation sites of the KRS1 gene were determined. Upstream from the TATA box, putative control elements corresponding to the concensus sequences for the RPG box and the general amino acid control system were identified. Evidence for transcriptional induction of the KRS1 gene via the general amino acid control system is presented.

Mapping of the constitutive lysyl-tRNA synthetase gene of Escherichia coli K-12.

The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis. Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.