Assessing incomplete deprotection of microarray oligonucleotides in situ

En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained

The Human Mitochondrial tRNAMet: Structure/Function Relationship of a Unique Modification in the Decoding of Unconventional Codons

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNAMetCAU, to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl-(A-) sites of the ribosome. The hmtRNAMetCAU has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f5C34), and a cytidine substituting for the invariant uridine at position 33 of the canonical “U-turn” in tRNAs. The structure of the tRNA's anticodon stem and loop domain (hmtASLMetCAU), determined by NMR restrained molecular modeling, revealed how the f5C34 modification facilitates the decoding of AUA at the P- and A-sites. The f5C34 defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASLMetCAU. The hmtASLMetCAU exhibited a “C-turn” conformation that has some characteristics of the U-turn motif. Codon binding studies with both E. coli and bovine mitochondrial ribosomes revealed that the f5C34 facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL's binding kinetics. Mitochondrial translation by many organisms including humans sometimes initiates with the universal isoleucine codons AUU and AUC. The f5C34 enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f5C34, expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria

Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome’s subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (Kds = 0.3 ± 0.1, 0.2 ± 0.2 and 5.4 ± 1.1 µM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (Kd = 2.6 ± 0.1 µM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides

Decoding the genome: a modi®ed view

ABSTRACT Transfer RNA's role in decoding the genome is critical to the accuracy and ef®ciency of protein synthesis. Though modi®ed nucleosides were identi®ed in RNA 50 years ago, only recently has their importance to tRNA's ability to decode cognate and wobble codons become apparent. RNA modi®ca-tions are ubiquitous. To date, some 100 different posttranslational modi®cations have been identi®ed. Modi®cations of tRNA are the most extensively investigated; however, many other RNAs have modi®ed nucleosides. The modi®cations that occur at the ®rst, or wobble position, of tRNA's anticodon and those 3¢-adjacent to the anticodon are of particular interest. The tRNAs most affected by individual and combinations of modi®cations respond to codons in mixed codon boxes where distinction of the third codon base is important for discriminating between the correct cognate or wobble codons and the incorrect near-cognate codons (