Communications t o the Editor 3971 exchange of protons and deuterons between aniline and D20. Therefore, no more than 20% of the observed reduction could have occurred via H2. If all of the reduction has occurred via Hg. there would have been observed 59% deuterium incorporation. We have also observed that there is no appreciable exchange between H2 and D20 under similar reaction conditions in the absence of nitrobenzene.

Proton nuclear magnetic resonance has been used to examine the effect of both noncomplementary and G.U oppositions in the duplexes formed by the synthetic pentaribonucleotides CpApApUpG, CpApUpUpG, CpApGpUpG, and CpApCpUpG. The lack of any sigmoidal behavior in the chemical shift vs. temperature plots of the base protons in the individual pentaribonucleotides indicates that duplexes with noncomplementary base oppositions of the type: formula: (see text), (where X = A, U, G, or C) do not form. Variable temperature spectra of the mixture of CpApGpUpG and CpApUpUpG were recorded over the range of 70--10 degrees C. The chemical shift vs. temperature plot of the purine aromatic protons displayed sigmoidal curves. This demonstrated both duplex formation and the presence of a G.U. base pair. The average Tm of the duplex was found to be 23.4 +/- 2.0 degrees C. This is similar to that of the duplex formed by CpApUpG (24.0 +/- 1.0 degrees C) but less than the Tm of the following duplexes: CpApApUpG:CpApUpUpG (Tm = 28.5 +/- 2.1 degrees C), CpApGpUpG:CpApCpUpG (Tm = 38.4 +/- 0.6 degrees C) and CpApUpApUpG (Tm = 41.5 +/- 1.1 degrees C). The G.U base pair has a Tm (20.0 degrees C) significantly lower than the rest of the duplex (24 +/- 1 degree C) and is a region of local instability within the double helix. This 1H NMR study is the first to investigate both the formation and relative stability of an internal G.U. base pair neighboring regular Watson--Crick base pairs.

Variable temperature lH n.m.r. spectroscopic studies have shown that the contribution of an internal G-U base pair approximates that of an A.U base pair in the stability of a short RNA helix, THE existence of G-U base pairs was first proposed in the wobble hypothesis of Crick1 to explain the codon-anticodon interactions between mRNA and tRNA. This base pair has since been included in the proposed secondary structures of several native R N A S ~~~~Q despite the fact that several physical studies on the existence of G-U base pairs were inconcl~sive.~ v6 Substantial evidence for the presence of a G-U interaction has recently been provided by the X-ray crystal structure of yeast tRNAPhe2 and the n.m.r. spectrum of polyd(GpT).' In order to study both the formation and relative stability of a G*U base pair in a short RNA helix flanked by normal Watson-Crick base-pairs, we have synthesized the pentaribonucleotides, CAGUG and CAUUG,? by the phosphotriester method.8 We have examined the duplex formation of these complementary sequences by lH n.m.r. spectroscopy.

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To examine the influence of bases contiguous to a starter codon, a rapid means of assembling biologically active regions corresponding to portions, or to analogues of portions of intercistronic regions is desirable. To do this, a chemical method for the specific insertion of 3′‐monophosphate groups on to chemically synthesized trinucleotides, tetranucleotides and hexanucleotides of defined sequence bearing different bases at the 3′ terminus has been devised. The method involved phosphorylation of blocked oligoribonucleotides synthesized by a phosphotriester method. The deblocked oligoribonucleotides were phosphorylated at the 5′ end with T4‐induced polynucleotide kinase. The products of this kinase reaction served as“donors” in RNA ligase reactions. The [32P]pC‐A‐U‐A‐U‐Gp, [32P]pA‐U‐Gp, [32P]pU‐A‐A, [32P]pA‐G‐G‐Ap, [32P]pC‐U‐U‐Ap and [32P]pU‐C‐C‐Up “donors” were used to synthesize A‐G‐G‐A[32P]pC‐A‐U‐A‐U‐Gp, U‐C‐C‐U[32P]pC‐A‐U‐A‐U‐Gp, U‐A‐A[32P]pA‐U‐G, A‐U‐G[32P]pU‐A‐A, U‐A‐A‐G[32P]pA‐G‐G‐Ap, U‐C‐C‐U[32P]pC‐U‐U‐Ap and A‐U‐U‐C[32P]pU‐C‐C‐Up indicating that the method functions with all bases. A‐U‐Gp, PA‐U‐Gp and pC‐A‐U‐A‐U‐Gp were isolated free of reactants and, along with PA‐U‐G, were all shown to promote the formation of translational initiation complexes. A‐G‐G‐A[32P]pC‐A‐U‐A‐U‐Gp, which corresponds to the 5′‐terminal portion of the intracistronic region of the maturation protein of bacteriophage Qβ, bound more efficiently to Escherichia coli ribosomes than the U‐C‐C‐U[32P]pC‐A‐U‐A‐U‐Gp, [32P]pC‐A‐U‐A‐U‐Gp, [32P]pA‐U‐G‐U‐A‐A or [32P]pU‐A‐A‐A‐U‐G controls. The results suggested that the content and number of residues at the 5′ terminus attached to A‐U‐G affect binding of oligonucleotides to ribosomes; purine nucleosides appear to be more effective than pyrimidine nucleosides in this regard.