Escherichia coli transcription factor NusG binds to 70S ribosomes

Abstract

Transcription and translation are coupled processes in bacteria. A role of transcription elongation cofactor NusG in coupling has been suggested by in vitro structural studies. NMR revealed association of the NusG carboxy-terminal domain with S10 (NusE), implying a direct role for NusG as a bridge linking RNAP and the lead ribosome. Here we present the first in vitro and in vivo evidence of full-length NusG association with mature 70S ribosomes. Binding did not require accessory factors in vitro. Mutating the NusG:S10 binding interface at NusG F165 or NusE M88 and D97 residues weakened NusG:S10 association in vivo and completely abolished it in vitro, supporting the specificity of this interaction. Mutations in the binding interface increased sensitivity to chloramphenicol. This phenotype was suppressed by rpoB*35, an RNAP mutation that reduces replisome-RNAP clashes. We propose that weakened NusG:S10 interaction leads to uncoupling when translation is inhibited, with resulting RNAP backtracking, replication blocks and formation of lethal DNA double-strand breaks.

Figure 1. Western blots and western dot blots of Superdex-200 column fractions

(Sections from separate blots were approximated to display all the required fractions. NusG/NusG F165A and ribosome/mutant ribosome preparations were used as controls on all western blots even if not displayed)

1. 70S ribosome elution profile. Fractions 11–23.

2. Wild type NusG elution profile. Fractions 11–23 and 31–41.

3. NusG and 70S (5:1) complex. Fractions 15–23 probed for ribosomes and NusG.

4. NusG F165A and 70S (5:1) combination. Fractions 15–23 and 31–36 probed for NusG.

5. NusG and mutant 70S (5:1) combination. Fractions 15–23 and 31–36 probed for NusG.

6. NusG F165A and mutant 70S (5:1) combination. Fractions 15–23 probed for ribosomes and NusG, fractions 31–36 probed for NusG.

7. NusG F165A and mutant 70S (10:1) combination. Fractions 15–23 and 31–36 probed for NusG.

Figure 2. NusG:ribosome association in vitro using pelleting assay

Supernatant (S), sucrose cushion (C) and pellet (P) probed for presence of NusG and ribosomes.

Figure 3. NusG:ribosome association in vivo

Equal quantities of crude ribosome preparations from MDS42, MDS42 nusE M88/D97A (11601) and MDS42 nusG F165A (10780) were probed for NusG (top panel) and ribosomes (lower panel). The slight misalignment of bands was caused by the presence of sucrose in the samples.

Figure 4. Mutations in the NusG:ribosome interface affect transcription-translation coupling in vivo

Colony-forming ability was assayed in the presence of 3 µg ml⁻¹ chloramphenicol (Cm) targeting translation elongation and 200 µg ml⁻¹ kasugamycin (Kas) targeting translation initiation. (A) The effect of nusE M88/D97A and other S10 mutations on strain growth in the presence of antibiotics. In every strain the nusE gene is linked to tetracycline-resistant Tn10. The strain order from top to bottom: 10401, KM821, 11601, KM820, 10400, KM812. (B) The effect of nusG F165A mutation on strain growth in the presence of antibiotics targeting translation. In all strains the nusG gene is linked to a kanamycin resistance cassette. The order of strains from top to bottom: KM848, KM852, KM850, KM854.