Abstract:Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined in in vitro translation extracts. Here, we investigate the potency, product distribution, and mechanism of various puromycin-oligonucleotide conjugates (1 to 44 nt with 39-puromycin) in a reticulocyte lysate cell-free translation system. Surprisingly, the potency decreases as the chain length of the oligonucleotide is increased in this series, and only very short puromycin conjugates function efficien… Show more
“…In our in vitro experiment, 6 mM puromycin inhibited approximately half of ORI5 substrate cleavage by MtMRP, and half of the ORI5 substrate cleavage activities by NuMRP were inhibited at 8 mM puromycin (Table 2). This is a concentration 1000-fold higher than that found to inhibit the ribosome (Starck and Roberts 2002), and equivalent to what was seen for the mouse RNase MRP and P enzymes (Potuschak et al 1993). This confirms puromycin as an inhibitor of RNase MRP but revealed no significant enzymatic differences between the two enzymes.…”
RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.
“…In our in vitro experiment, 6 mM puromycin inhibited approximately half of ORI5 substrate cleavage by MtMRP, and half of the ORI5 substrate cleavage activities by NuMRP were inhibited at 8 mM puromycin (Table 2). This is a concentration 1000-fold higher than that found to inhibit the ribosome (Starck and Roberts 2002), and equivalent to what was seen for the mouse RNase MRP and P enzymes (Potuschak et al 1993). This confirms puromycin as an inhibitor of RNase MRP but revealed no significant enzymatic differences between the two enzymes.…”
RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.
“…This observation is consistent with the molecules
being stable in FBS over the 4 h incubation time. On the other hand,
in vitro translated Pep1 (natural carboxy C-terminus and therefore
sensitive to carboxypeptidase degradation) 24 shows a distinctly lower signal in FBS versus assay buffer, consistent
with peptide degradation. Overall, our results show that C-terminally
blocked peptides (fusions and amidated peptides) show excellent performance
in 1% FBS and argue that these reagents can be used in complex media-containing
proteases and peptidases.Figure 5Peptides have modification-dependent stability in serum.…”
A major benefit of
proteomic and genomic data is the potential
for developing thousands of novel diagnostic and analytical tests
of cells, tissues, and clinical samples. Monoclonal antibody technologies,
phage display and mRNA display, are methods that could be used to
generate affinity ligands against each member of the proteome. Increasingly,
the challenge is not ligand generation, rather the analysis and affinity
rank-ordering of the many ligands generated by these methods. Here,
we developed a quantitative method to analyze protein interactions
using in vitro translated ligands. In this assay, in vitro translated
ligands generate a signal by simultaneously binding to a target immobilized
on a magnetic bead and to a sensor surface in a commercial acoustic
sensing device. We then normalize the binding of each ligand with
its relative translation efficiency in order to rank-order the different
ligands. We demonstrate the method with peptides directed against
the cancer marker Bcl-xL. Our method has 4- to 10-fold
higher sensitivity, using 100-fold less protein and 5-fold less antibody
per sample, as compared directly with ELISA. Additionally, all analysis
can be conducted in complex mixtures at physiological ionic strength.
Lastly, we demonstrate the ability to use peptides as ultrahigh affinity
reagents that function in complex matrices, as would be needed in
diagnostic applications.
“…(Fig. 2B) It has been shown that puromycin substituted at the 5 0 OH has decreased efficacy as a translation inhibitor, so a deoxycytidine (dC) was used to link fluorescent dyes (Cy5 and fluorescein) to puromycin (Starck and Roberts 2002;Starck et al 2004). Fluorescein-dC-puromycin (F2P) has been used to measure global protein synthesis in the dendrites of rat hippocampal neurons.…”
The regulation of translation provides a mechanism to control not only the abundance of proteins, but also the precise time and subcellular location that they are synthesized. Much of what is known concerning the molecular basis for translational control has been gleaned from experiments (e.g., luciferase assays and polysome analysis) that measure average changes in the protein synthesis of a population of cells, however, mechanistic insights can be obscured in ensemble measurements. The development of fluorescent microscopy techniques and reagents has allowed translation to be studied within its cellular context. Here we highlight recent methodologies that can be used to study global changes in protein synthesis or regulation of specific mRNAs in single cells. Imaging of translation has provided direct evidence for local translation of mRNAs at synapses in neurons and will become an important tool for studying translational control.
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