To save content items to your account,
please confirm that you agree to abide by our usage policies.
If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your account.
Find out more about saving content to .
To save content items to your Kindle, first ensure firstname.lastname@example.org
is added to your Approved Personal Document E-mail List under your Personal Document Settings
on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part
of your Kindle email address below.
Find out more about saving to your Kindle.
Note you can select to save to either the @free.kindle.com or @kindle.com variations.
‘@free.kindle.com’ emails are free but can only be saved to your device when it is connected to wi-fi.
‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.
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”
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
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
Email your librarian or administrator to recommend adding this to your organisation's collection.