Characterization of translational initiation sites in E. coli.

We characterize the Shine and Dalgarno sequence of 124 known gene beginnings. This information is used to make "rules" which help distinguish gene beginning from other sites in a library of over 78,000 bases of mRNA. Gene beginnings are found to have information besides the initiation codon and Shine and Dalgarno sequence which can be used to make better "rules".     

Multiply branched replicative intermediates in E. coli and bacteriophage lambda

Summary Multiple branched DNA fragments present in a fast sedimenting complex comprising a minute fraction of the E. coli genome have been isolated. Similar structures were also observed among bacteriophage DNA replicative intermediates after infection of synchronized E. coli cells. These structures were found to be associated with the amino acid and thymidine starvation steps required for synchronization and originate either by initiation from secondary sites or by snap-back of daughter strands containing substantial single stranded regions in the vicinity of the growing point.     

Topography of the E site on the Escherichia coli ribosome.

Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucleotides. We have located the two extremities of the E site-bound tRNA on the ribosomal subunits according to the positions of L33, S11 and the 3' end of 16S rRNA defined by immune electron microscopy. Our results demonstrate conclusively that the E site is topographically distinct from either the P site or the A site, and that it is located alongside the P site as expected for the tRNA exit site.     

An initiation factor causing dissociation of E. coli ribosomes

Purification of crude initiation factors, essential for polypeptide synthesis in cell-free systems of E. coli, yielded a fraction DF which causes dissociation of 70 S ribosomes. Its stoichiometric action on 70 S ribosomes is antagonized by increasing Mg(2+) concentrations but not by the addition of 30 S and 50 S subunits washed with high salt concentration. GTP did not stimulate this dissociating action. 2 &mgr;g of our most purified preparation caused 100% dissociation of 100 &mgr;g of 70 S ribosomes without added GTP. DF-induced dissociation is a very rapid process at 37 degrees C and is temperature-dependent in the range of 0 degrees -37 degrees C. DF, which is thermolabile factor, is much less or not effective with complexed 70 S ribosomes bearing peptidyl-tRNA and mRNA.