David S. McPheeters

We have developed a new technique, called 'toeprinting,' which has allowed a study of the tRNA-binding properties of Escherichia coli translation initiation complexes. In response to natural mRNAs, the initiator tRNA and a variety of elongator tRNAs bind to the same tRNA-binding site on the 30S ribosomal subunit as long as a cognate codon is present near the Shine and Dalgarno sequence. The selection of the initiator tRNA in 30S initiation complexes is accomplished by initiation factors IF2 and IF3. 70S ribosomes accept both initiator tRNA and elongator tRNAs on natural mRNAs, much like 30S ribosomal subunits; IF3 and IF2 do not, however, select the initiator tRNA on 70S initiation complexes unless the initiation factor IF1 is present.

U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the T7U6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.

The bacteriophage T4 lysozyme gene is transcribed at early and late times after infection of E. coli, but the early mRNA is not translated. DNA sequence analysis and mapping of the 5' ends of the lysozyme transcripts produced at different times after T4 infection show that the early mRNA is initiated some distance upstream from the gene. The early mRNA is not translated because of a stable secondary structure which blocks the translational initiation site. The stable RNA structure has been demonstrated by nuclease protection in vivo. After DNA replication begins, two late promoters are activated; the late transcripts are initiated at sites such that the secondary structure can not form, and translation of the late messages occurs.