Despite the well-characterised mechanisms of amino acids (AA) regulation of milk protein synthesis in mammary glands (MG), the underlying specific AA regulatory machinery in bovine MG remains further elucidated. As methionine (Met) is one of the most important essential and limiting AA for dairy cows, it is crucial to expand how Met exerts its regulatory effects on dairy milk protein synthesis. Our previous work detected the potential regulatory role of seryl-tRNA synthetase (SARS) in essential AA (EAA)-stimulated bovine casein synthesis. Here, we investigated whether and how SARS participates in Met stimulation of casein production in bovine mammary epithelial cells (BMEC). With or without RNA interference against SARS, BMEC were treated with the medium in the absence (containing all other EAA and devoid of Met alone)/presence (containing 0·6 mm of Met in the medium devoid of Met alone) of Met. The protein abundance of β-casein and members of the mammalian target of rapamycin (mTOR) and general control nonderepressible 2 (GCN2) pathways was determined by immunoblot assay after 6 h treatment, the cell viability and cell cycle progression were determined by cell counting and propidium iodide-staining assay after 24 h treatment, and protein turnover was determined by l-[ring-3H5]phenylalanine isotope tracing assay after 48 h treatment. In the absence of Met, there was a general reduction in cell viability, total protein synthesis and β-casein production; in contrast, total protein degradation was enhanced. SARS knockdown strengthened these changes. Finally, SARS may work to promote Met-stimulated β-casein synthesis via affecting mTOR and GCN2 routes in BMEC.

Preparation of large quantities of RNA molecules of a defined sequence is a prerequisite for biophysical analysis, and is particularly important to the determination of high-resolution structure by X-ray crystallography. We describe improved methods for the production of multimilligram quantities of homogeneous tRNAs, using a combination of chemical synthesis and enzymatic approaches. Transfer RNA half-molecules with a break in the anticodon loop were chemically synthesized on a preparative scale, ligated enzymatically, and cocrystallized with an aminoacyl-tRNA synthetase, yielding crystals diffracting to 2.4 Å resolution. Multimilligram quantities of tRNAs with greatly reduced 3′ heterogeneity were also produced via transcription by T7 RNA polymerase, utilizing chemically modified DNA half-molecule templates. This latter approach eliminates the need for large-scale plasmid preparations, and yields synthetase cocrystals diffracting to 2.3 Å resolution at much lower RNA:protein stoichiometries than previously required. These two approaches developed for a tRNA–synthetase complex permit the detailed structural study of “atomic-group” mutants.

2′-Deoxynucleoside 5′-α-thiotriphosphates have been incorporated randomly, replacing any of the four nucleotides separately and at a low level in Escherichia coli tRNAAsp transcripts. After some tRNAs were charged with the cognate aminoacyl-tRNA synthetase and biotinylated, charged and uncharged tRNAs were separated by binding to Streptavidin. A comparison of the iodine cleavage pattern of charged and uncharged tRNAs indicated positions of 2′-deoxy-phosphorothioate interference with charging. To separate the 2′-deoxy from the phosphorothioate effect, the same sequence of reactions was performed with the corresponding NTPαS. Several positions were identified with a 2′-deoxy or a phosphorothioate effect. tRNAs with single deoxy substitutions at the identified positions were prepared by enzymatic ligation of chemically synthesized halves. The kinetics of charging these tRNAs were determined. The 2′-deoxy effects identified by the interference assay were confirmed, showing a reduction in charging efficiency of between 2.5–6-fold, except for the terminal A76 with a 25-fold reduction. Inspection of the X-ray structure of the tRNA-synthetase complex showed consistency of most of these findings. Critical 2′-deoxy groups are localized mainly on the proposed contact surface with the synthetase or at the interface of the two tRNA domains. The same overall picture emerged for critical phosphorothioates. With the exception of 2′-deoxy-adenosine-containing tRNAs, multiple 2′-deoxy-substituted tRNAs, prepared by ligation of halves, showed a much larger reduction in charging efficiency than the mono-substituted tRNAs, indicating an additive effect.

The use of T7 RNA polymerase to prepare large quantities of RNA of a particular sequence has greatly facilitated the study of both the structure and function of RNA. Generally, it has been believed that the products of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5′ end sequence and found that, for those molecules that begin with multiple, consecutive guanosines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5′ end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 5′ ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 5′ end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5′ end heterogeneity in combination with the 3′ end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification.

An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3′-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive.