Ribosome recycling factor and release factor 3 action promotes TnaC-peptidyl-tRNA Dropoff and relieves ribosome stalling during tryptophan induction of tna operon expression in Escherichia coli

Abstract

Upon tryptophan induction of tna operon expression in Escherichia coli, the leader peptidyl-tRNA, TnaC-tRNA(2)(Pro), resists cleavage, resulting in ribosome stalling at the tnaC stop codon. This stalled ribosome blocks Rho factor binding and action, preventing transcription termination in the tna operon's leader region. Plasmid-mediated overexpression of tnaC was previously shown to inhibit cell growth by reducing uncharged tRNA(2)(Pro) availability. Which factors relieve ribosome stalling, facilitate TnaC-tRNA(2)(Pro) cleavage, and relieve growth inhibition were addressed in the current study. In strains containing the chromosomal tna operon and lacking a tnaC plasmid, the overproduction of ribosome recycling factor (RRF) and release factor 3 (RF3) reduced tna operon expression. Their overproduction in vivo also increased the rate of cleavage of TnaC-tRNA(2)(Pro), relieving the growth inhibition associated with plasmid-mediated tnaC overexpression. The overproduction of elongation factor G or initiation factor 3 did not have comparable effects, and tmRNA was incapable of attacking TnaC-tRNA(2)(Pro) in stalled ribosome complexes. The stability of TnaC-tRNA(2)(Pro) was increased appreciably in strains deficient in RRF and RF3 or deficient in peptidyl-tRNA hydrolase. These findings reveal the existence of a natural mechanism whereby an amino acid, tryptophan, binds to ribosomes that have just completed the synthesis of TnaC-tRNA(2)(Pro). Bound tryptophan inhibits RF2-mediated cleavage of TnaC-tRNA(2)(Pro), resulting in the stalling of the ribosome translating tnaC mRNA. This stalling results in increased transcription of the structural genes of the tna operon. RRF and RF3 then bind to this stalled ribosome complex and slowly release TnaC-tRNA(2)(Pro). This release allows ribosome recycling and permits the cleavage of TnaC-tRNA(2)(Pro) by peptidyl-tRNA hydrolase.

FIG. 1.

Overproduction of RRF and RF3 relieves the growth inhibition caused by tnaC overexpression. (A) Schematic representation of the organization of plasmids pTnaC and pTnaC-RRF. Both plasmids are pUC18 derivatives and contain the tna operon promoter, the tnaC open reading frame, the noncoding region located between tnaC and tnaA genes, and an added rpoBC terminator. In preparing pTnaC-RRF, a fragment containing the RRF coding region and its own promoter from plasmid pRR1 was subcloned into plasmid pTnaC immediately following the rpoBC terminator. (B) Overproduction of both RRF and RF3 relieves the growth inhibition caused by tnaC overexpression by pTnaC in the presence of tryptophan. E. coli cells bearing the different plasmids listed in the figure (Table 1) were diluted from cultures grown overnight and were grown with shaking at 37°C in minimal medium supplemented with 0.05% acid-hydrolyzed casein and 0.2% glycerol with (+) or without (−) 100 μg/ml tryptophan (Trp). Cell growth was monitored at an OD₆₀₀. A total of 10 mM IPTG (+) was added at time zero for RF3 overproduction. Four independent growth experiments were performed. The growth curves for the different strains grown with or without added tryptophan were all similar to those shown in this figure.

FIG. 2.

tnaC overexpression does not affect the half-life of TnaC- in vivo. Strain SVS1144 (A) or SVS144 harboring plasmid pTnaC (B) was grown at 37°C in minimal medium supplemented with 0.05% acid-hydrolyzed casein and 0.2% glycerol with (+) or without (−) the addition of 100 μg/ml tryptophan (Trp). The OD₆₀₀ at the time of glucose addition (final concentration of 1.0%) to each culture was 0.6 to 0.8. Samples were taken at the indicated times, harvested by centrifugation, and disrupted by sonication, and equivalent amounts of total protein were electrophoresed on a 10% Tricine-sodium dodecyl sulfate acrylamide gel. Northern blotting was then performed to quantify the level of TnaC- (TC-tRNAP). The TnaC- band was detected at 26 kDa. The  (tRNAP) molecule was also detected at 14 kDa; however, measurements of  levels were not accurate quantitatively (10). The measured half-life (t1/2) of TC-tRNAP, calculated using the curves in C, is given to the right of each set of data. Little or no TnaC- was detected in the samples from uninduced cultures. The percentage of TnaC- relative to the level at 0 min was calculated by dividing the densitometry units obtained from the TnaC- band in each lane by the units obtained for the TnaC- band in the 0-min lane. Note that there is less TnaC- in the 0-min sample from chromosomal tnaC expression (compare +Trp TC-tRNAP values in A with those in B). (C) Plot of TnaC- decay curves based on the data shown in A and B. Plots are not shown for the cultures grown without tryptophan. At least three independent experiments were performed, and the TnaC- values obtained for any strain and condition varied by less than 20%.

FIG. 3.

Overproduction of RRF and RF3 reduces the half-life of TnaC- in vivo. (A) Strains bearing the indicated plasmid or plasmids were grown at 30°C or 37°C in minimal medium supplemented with 0.05% acid-hydrolyzed casein and 0.2% glycerol with the addition of 100 μg/ml tryptophan (Trp). In strain SVS1144(prfCΔ2::kan), the prfC gene was deleted. Strain SVS1144[frr(Ts)] produces an RRF protein that is temperature sensitive. The frr(Ts) allele was also present in SVS1144[frr(Ts) prfCΔ2::kan]. Strains with plasmid pIQ-RF3 were grown with 10 mM IPTG, which induces RF3 production. Plasmid pTnaC-RRF was used to overexpress tnaC and overproduce RRF. Strain SVS1144[frr(Ts)] pTnaC was grown initially at 30°C and then shifted to 37°C for 1 h. Presumably, most of its RRF protein would be inactivated during growth at 37°C. The OD₆₀₀ at the time of glucose addition (final concentration of 1.0%) to each culture was 0.6 to 0.8. Samples were taken and sonicated, and Northern blot assays performed to detect TnaC- (TC-tRNAP) and  (tRNAP), as indicated in the legend of Fig. 2. The half-life (t1/2) of TnaC-, calculated using the curve in C, is shown to the right of each set of data. The relative TnaC- level at 0 min is shown to the left of each panel. These values were calculated by dividing the densitometry units obtained for the TnaC- band in each 0-min lane by the densitometry units obtained for the TnaC- band in the 0-min lane for the control SVS1144(pTnaC) culture. (B) The TnaC- decay curves are based on the percentage of TnaC- detected relative to the 0-min value, as indicated in Fig. 2. At least three independent experiments were performed, and the TnaC- values obtained for any strain and condition varied by less than 20%.

FIG. 4.

Pth (peptidyl-tRNA hydrolase) is responsible for the cleavage of TnaC- in vivo. Cultures of strains C600 (wild type) and C600[pth(Ts)] (temperature-sensitive Pth) either without (A) or with (B) plasmid pTnaC (+pTnaC) were grown in LB medium {C600[pth(Ts)]} at 30°C to an OD₆₀₀ of 0.8. They were then shifted to 42°C and incubated for 1 h. Glucose was then added to 1%, and samples were harvested at the indicated times. Cultures of wild-type (wt) strain C600 grown at 42°C were used as controls. Northern blot assays were performed to detect TnaC- (TC-tRNAP) and  (tRNAP), and the percentage of TnaC- detected relative to the 0-min value was calculated as described in the legend of Fig. 2. The half-life (t1/2) of TnaC-, calculated using the curve in C, is shown to the right of each set of data in A and B. (C) TnaC- decay curves based on the data in A and B. At least two independent experiments were performed, and the TnaC- values obtained for any strain and condition varied by less than 15%.