Inhibition of a transcriptional pause by RNA anchoring to RNA polymerase.

We describe a mechanism by which nascent RNA inhibits transcriptional pausing. PutL RNA of bacteriophage HK022 suppresses transcription termination at downstream terminators and pausing within a nearby U-rich sequence. In vitro transcription and footprinting assays reveal that this pausing results from backtracking of RNA polymerase and that binding of nascent putL RNA to polymerase limits backtracking by restricting re-entry of the transcript into the RNA exit channel. The restriction is local and relaxes as the transcript elongates. Our results suggest that putL RNA binds to the surface of polymerase close to the RNA exit channel, a region that includes amino acid residues important for antitermination. Although binding is essential for antipausing and antitermination, these two activities of put differ: antipausing is limited to the immediate vicinity of the putL site, but antitermination is not. We propose that RNA anchoring to the elongation complex is a widespread mechanism of pause regulation.

The mutation that makes Escherichia coli resistant to lambda P gene-mediated host lethality is located within the DNA initiator Gene dnaA of the bacterium.

Earlier, we reported that the bacteriophage lambda P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this lambda P gene-mediated lethality. In this paper, we show that under the lambda P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94 % linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from lambda P gene-mediated killing and complements E. coli dnaAts46 at 42 degrees C. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to lambda P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.

Protein arginine methyltransferase I: substrate specificity and role in hnRNP assembly.

Prmt1, the major protein arginine methyltransferase in mammalian cells, has been implicated in signal transduction, transcriptional control, and protein trafficking. In the present study, mouse embryonic stem cells homozygous for an essentially null mutation in the Prmt1 gene were used to examine Prmt1 activity and substrate specificity, which by several criteria appeared to be highly specific. First, other methyltransferases did not substitute for the loss of Prmt1 activity. Second, almost all proteins modified by recombinant Prmt1 in vitro were authentic substrates, i.e., proteins rendered hypomethylated by Prmt1 gene disruption. Finally, Prmt1 did not modify the substrates of other methyltransferases from cells treated with methyltransferase inhibitors. Recombinant proteins corresponding to two splice-variants, Prmt1(353) and Prmt1(371), methylated different, proteins in vitro, providing the first evidence for functional differences between the two isoforms. However, the differences in substrate specificity were lost by the addition of an N-terminal His(6) tag. Loss of Prmt1 activity (and hypomethylation of hnRNPs) has no obvious effect on the formation or composition of hnRNP complexes. Finally, methylation of the most abundant Prmt1 substrates appeared to be extensive and constitutive throughout the cell cycle, suggesting the modification does not modulate protein function under normal growth conditions.