Requirement for NusG for Transcription Antitermination In Vivo by the λ N Protein

Abstract

Transcription antitermination by the bacteriophage N protein is stimulated in vitro by the Escherichia coli NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription antitermination in intact cells. (8,20), is an abundant transcription elongation factor that modulates Rho-dependent transcription termination (22,24,25). Rhodependent termination is inhibited by depletion or overexpression of NusG (2, 24). Affinity chromatography experiments showed that NusG associates with Rho or core RNA polymerase (19). It was proposed that NusG facilitates termination by aiding in the recruitment of Rho to the transcription complex (1,18,21,22,24). NusG, a 21-kDa essential protein in Escherichia coliIn vitro studies identified NusG as an E. coli protein that influences transcription antitermination mediated by the N protein of phage (18,20). Transcription of the delayed early genes requires RNA polymerase to read through a number of transcription terminators. N, in complex with the E. coli Nus factors, modifies RNA polymerase at specific RNA sites, NUT, to a form that overrides downstream transcription terminators ( Fig. 1) (7,11,12,16,23,27).Nus factors were first identified by mutations in E. coli that influence N action. Mutations in nusA (nusA1) (9), nusB (a number of mutations) (10, 17), and rpsJ (nusE71) (14) reduce N activity, particularly at higher temperatures. In contrast, the nusB101 mutation suppresses this effect of the nusA1 and nusE71 alleles on N action (26).The only genetic evidence for a role of NusG in N antitermination was the isolation of nusG4, which has a suppressor phenotype identical to that of the nusB101 allele (25). However, selection for nus mutations that reduce N action did not yield nusG mutants. Furthermore, in contrast to its function in vitro, in vivo studies provided evidence that NusG was not required for effective N-mediated transcription antitermination. The in vivo studies employed a strain bearing a lacZ reporter fused downstream of the p L promoter, nutL, and transcription terminators. Because of the terminators, expression of the lacZ reporter is N dependent. NusG was expressed from a plasmid with a temperature-sensitive replicon. NusG depletion was accomplished by shifting the strain from 32 to 42°C. N was expressed from a plasmid under the control of the lac promoter. The experimental design was to supply N after depleting the bacteria of NusG (see below for the method of depletion) by treating the bacteria with IPTG (isopropyl-␤-Dthiogalactopyranoside). Depleting this strain of NusG did not reduce lacZ expression, suggesting that N-mediated antitermination did not require NusG (24). However, more recent experiments show that this result can be explained on the basis of leaky expression of N prior to the depletion of NusG (data not shown). We were concerned because the plasmid supplying N was present during the entire time of growth at 42°C. Since the p L -nutL-t L1 -lacZ fusion ...

Essentiality of Ribosomal and Transcription Antitermination Proteins Analyzed by Systematic Gene Replacement in Escherichia coli

“…The NusA and NusD/Rho functions were previously suspected to be essential for bacterial growth, since conditional lethal mutants of these genes had been isolated (13,17,39,56). A nusG knockout was shown to be nonviable (72), while a nusA knockout could be obtained only in the presence of attenuating rho mutations (71). At the same time, rho (45) and nusG (27) been reported as viable.…”

Host Factor Titration by Chromosomal R-loops as a Mechanism for Runaway Plasmid Replication in Transcription Termination-defective Mutants of Escherichia coli

“…22,41,42 The recessive nusG-G146D mutation is the first example of a viable nusG allele that confers a transcription termination-defective phenotype. The mutation phenocopies a defective rho allele (rho-A243E) in conferring phage P2 resistance and relief of transcriptional polarity at each of four widely separated genetic loci (Figure 1), implying a global deficiency of Rho-dependent transcription termination in the nusG mutant.…”

In Vivo Effect of NusB and NusG on rRNA Transcription Antitermination

“…These investigators suggested that the very rapid transcription of rRNA by T7 RNA polymerase uncouples the delicate relationship between transcription and ribosomal assembly (18). This result raises the possibility that the twofold antiterminator-dependent increase in the transcription elongation rate is necessary for the proper folding of the rRNA and the subsequent assembly of the ribosomal subunits (38,40). c cat/bla mRNA ratio normalized to that for pSL102.…”