N-Acetylvalyl-tRNAYl (AcVal-tRNA 1) was bound to the P site of uniformly 32P-labeled 70S ribosomes from Escherichia coli and crosslinked to 16S RNA in the 30S ribosomal subunit by irradiation with light of300-400 nm. To identify the crosslinked nucleotide in 16S RNA, AcVal-tRNAY. l-16S [32P]RNA was digested completely with RNase TI and the band containing the covalently attached oligonucleotides from tRNA and rRNA was isolated by polyacrylamide gel electrophoresis. The crosslinked oligonucleotide, and the 32P-labeled rRNA moiety released from it by photoreversal of the crosslink at 254 nm, were then analyzed by secondary hydrolysis with pancreatic RNase A and RNase U2. The oligonucleotide derived from 16S RNA was found to be the evolutionarily conserved sequence, U-A-C-A-C-A-C-C-G1401, and the nucleotide crosslinked to tRNAI , C14W. The identity of the covalently attached residue in the tRNA was established by using AcVal-tRNAlv-16S RNA prepared from unlabeled ribosomes.This complex was digested to completion with RNase T1 and the resulting RNA fragments were labeled at the 3' end with [5'-32P]pCp. The crosslinked Ti oligonucleotide isolated from the mixture yielded one major end-labeled component upon photoreversal. Chemical sequence analysis demonstrated that this product was derived from the anticodon-containing pentadecanucleo-

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Despite well-documented improvement in attention deficit hyperactivity disorder (ADHD) in youths with the available single and combined pharmacologic agents, a number of youths remain with residual symptomatology causing impairment in multiple domains. Recent work has suggested a potential cognitive-enhancing role of cholinergic agents in ADHD. We describe five cases of ADHD youths aged 8-17 years being treated for ADHD with the acetylcholinesterase inhibitor donepezil (Aricept), all of whom demonstrated improvement.

A mutation affording low levels of erythromycin resistance has been obtained by in vitro hydroxylamine mutagenesis of a cloned ribosomal RNA operon from Escherichia coli. The site of the mutational event responsible for antibiotic resistance was localized to the gene region encoding domain II of23S rRNA by replacement of restriction fragments in the wild-type plasmid by corresponding fragments from the mutant plasmid. DNA sequencing showed that positions 1219-1230 of the 23S rRNA gene are deleted in the mutant. Since all previously characterized rRNA mutations conferring resistance to erythromycin show changes exclusively in domain V, our present findings provide direct evidence for functional interaction between domains II and V of 23S rRNA.Efforts to understand the role ofrRNA in translation have led to the identification ofseveral putative functional sites in 16S-and 23S-like rRNAs (reviewed in ref. 1). Our present knowledge of the structural organization of these sites is limited to a description of their secondary structures and, in some cases, their locations relative to the electron microscopic models for the ribosomal subunits (reviewed in ref.2). Deeper insight into the mechanisms of rRNA function would undoubtedly result from information relevant to the three-dimensional arrangement of these sites.The secondary structure models for the rRNAs suggest that they are organized into discrete structural domains (1). The strong phylogenetic conservation ofthese domains raises the question as to whether this has some important functional basis, as has been established for many proteins. Specifically, is it possible that there is functional interaction between rRNA domains during the course of protein synthesis? One of the most direct approaches, chemical crosslinking, has provided evidence for close proximity between specific regions of different rRNA domains (reviewed in ref.3).Genetic approaches, which are potentially among the most powerful in exploring the functional and structural organization of rRNA, have yet to be extensively exploited in this context. To a great extent, this is because of the difficulty of performing genetic analysis on an essential gene that is present in seven copies in the genome ofEscherichia coli, the reference organism for study of ribosomes. This problem has been alleviated by using rRNA operons that have been cloned in high-copy-number plasmids. This approach has permitted isolation of rRNA mutants from E. coli that show observable phenotypic changes (4), including antibiotic resistance (5).In this paper we describe an erythromycin-resistant mutant, obtained by random in vitro mutagenesis of a cloned rRNA operon. The site of mutation lies in domain II of 235 rRNA, remote from all previously identified erythromycinresistant rRNA mutations, which have been localized to a small region in domain V (6, 7). We take this to be direct evidence for functional interaction between two different domains in a rRNA. MATERIALS AND METHODSCell Strains and Medium. E. coli strain DH1 [F...