Chemically modified nucleotide analogs have gained widespread popularity for probing structure–function relationships. Among the modifications that were incorporated into RNAs for assessing the role of individual functional groups, the phenyl nucleotide has displayed surprising effects both in the contexts of the hammerhead ribozyme and pre-mRNA splicing. To examine the conformational properties of this hydrophobic base analog, we determined the crystal structure of an RNA double helix with incorporated phenyl ribonucleotides at 1.97 Å resolution. In the structure, phenyl residues are engaged in self-pairing and their arrangements suggest energetically favorable stacking interactions with 3′-adjacent guanines. The presence of the phenyl rings in the center of the duplex results in only moderate changes of the helical geometry. This finding is in line with those of earlier experiments that showed the phenyl analog to be a remarkably good mimetic of natural base function. Because the stacking interactions displayed by phenyl residues appear to be similar to those for natural bases, reduced conformational restriction due to the lack of hydrogen bonds with phenyl as well as alterations in its solvent structure may be the main causes of the activity changes with phenyl-modified RNAs.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 ± 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.

Oxygen evolution was measured from mesophyll tissues in spinach leaves using a photoacoustic technique. The photosynthetic capacity of individual cell layers was measured by directing microscopic beams of light, 40 μm wide, to cells exposed within a leaf cross section. The resulting profile for oxygen-evolution potential was relatively flat, indicating a uniform capacity for photosynthesis in leaf mesophyll tissues. Two experimental approaches were used to estimate the photosynthetic performance of individual mesophyll cell layers when white light was applied to the adaxial leaf surface. These experiments indicated that oxygen was produced relatively uniformly across the mesophyll and that oxygen evolution increased with irradiance of the white light applied to the leaf surface. The measured profiles for oxygen evolution and capacity are flatter than previous measurements of profiles of fixed carbon and estimates of profiles for absorbed light within spinach leaves.