SuhB Associates with Nus Factors To Facilitate 30S Ribosome Biogenesis in Escherichia coli

Abstract

A complex of highly conserved proteins consisting of NusB, NusE, NusA, and NusG is required for robust expression of rRNA in Escherichia coli. This complex is proposed to prevent Rho-dependent transcription termination by a process known as "antitermination." The mechanism of this antitermination in rRNA is poorly understood but requires association of NusB and NusE with a specific RNA sequence in rRNA known as BoxA. Here, we identify a novel member of the rRNA antitermination machinery: the inositol monophosphatase SuhB. We show that SuhB associates with elongating RNA polymerase (RNAP) at rRNA in a NusB-dependent manner. Although we show that SuhB is required for BoxA-mediated antitermination in a reporter system, our data indicate that the major function of the NusB/E/A/G/SuhB complex is not to prevent Rho-dependent termination of rRNA but rather to promote correct rRNA maturation. This occurs through formation of a SuhB-mediated loop between NusB/E/BoxA and RNAP/NusA/G. Thus, we have reassigned the function of these proteins at rRNA and identified another key player in this complex.

Importance: As RNA polymerase transcribes the rRNA operons in E. coli, it complexes with a set of proteins called Nus that confer enhanced rates of transcription elongation, correct folding of rRNA, and rRNA assembly with ribosomal proteins to generate a fully functional ribosome. Four Nus proteins were previously known, NusA, NusB, NusE, and NusG; here, we discover and describe a fifth, SuhB, that is an essential component of this complex. We demonstrate that the main function of this SuhB-containing complex is not to prevent premature transcription termination within the rRNA operon, as had been long claimed, but to enable rRNA maturation and a functional ribosome fully competent for translation.

FIG 1

SuhB is functionally related to Nus factors. (A) Growth phenotypes of single mutants of mfd, nusA, nusB, rho, and rfaH in the W3110 wild-type (wt) background compared to the phenotypes for strains with the same mutations combined with deletion of suhB. Cells were restreaked onto LB medium and grown overnight at 37°C. (B) RNA-seq comparison of wild-type MG1655 and an isogenic ΔnusB strain. Each dot represents an annotated gene. Values on each axis indicate the relative number of sequence reads mapping to a given gene (plotted on a log scale), with values for the wild type plotted on the x axis and values for the ΔnusB mutant plotted on the y axis. Data points shown in black indicate genes for which we detected a significant difference (FDR, <0.05) of >2-fold between the wild-type and ΔnusB strain. (C and D) Equivalent comparisons for wild-type and ΔsuhB strains (C) and ΔnusB and ΔsuhB strains (D).

FIG 2

SuhB is required for BoxA-mediated antitermination. (A) Plasmids used for the antitermination reporter assay. pSL102 contains cat under control of a constitutive promoter. pSL103 is a derivative of pSL102 that includes a 567-bp noncoding sequence in the 5′-UTR of cat. pSL115 is a derivative of pSL103 that includes a BoxA-containing sequence from the rRNA leader in the 5′-UTR. All plasmids contain bla, which served as a normalization control. (B) qRT-PCR was used to determine the levels of cat mRNA relative to bla mRNA for each of the three plasmids in wild-type, ΔnusB, and ΔsuhB cells, as indicated. Data represent an average of three independent, biological replicates. Error bars indicate 1 standard deviation above and below the mean. (C) qRT-PCR was used to determine the levels of cat mRNA relative to bla mRNA for pSL115 in wild-type, ΔnusB, and ΔsuhB cells that contained either empty vector (pBAD18) or expressed SuhB from a plasmid (pBAD18-suhB).

FIG 3

SuhB is recruited to elongating RNAP in a boxA-dependent, NusB-dependent manner. (A) The level of SuhB-FLAG₃ and RNAP (β) association across rRNA loci was measured using ChIP-qPCR for wild-type (wt; SuhB-FLAG) and ΔnusB (SuhB-FLAG ΔnusB) cells. As a control, the experiment was performed with untagged, wild-type MG1655. Data points indicate the level of ChIP signal using the FLAG antibody (i.e., association of SuhB or the negative control; α-FLAG), normalized to that of RNAP β. The position of each data point on the x axis indicates the center of the PCR amplicon. A schematic of an rRNA locus is drawn to scale, aligned with the data. The boxA sequence is represented by a red rectangle. (B) The level of SuhB-FLAG₃ and RNAP (β) association across lacZYA was measured using ChIP-qPCR for wild-type cells (SuhB-FLAG wt lacZYA) and cells in which a boxA-containing sequence had been inserted in the lacZYA 5′-UTR (SuhB-FLAG, boxA-lacZYA). Data points indicate the level of ChIP signal detected using the FLAG antibody (i.e., association of SuhB or the negative control), normalized to that of RNAP β. The position of each data point on the x axis indicates the center of the PCR amplicon. Note that we grew cells in the presence of the inducer isopropyl-β-d-thiogalactopyranoside, but lacZYA was constitutively transcribed in the boxA-lacZYA strain because the boxA insertion interrupts a binding site for the LacI repressor (65). A schematic of the lacZYA operon is drawn to scale, aligned with the data. The inserted boxA sequence is represented by a red rectangle in the 5′-UTR. Data represent averages of three independent, biological replicates. Error bars indicate 1 standard deviation above and below the mean.

FIG 4

Limited role for NusB and SuhB in rRNA antitermination. ChIP-qPCR was used to measure the association of RNAP (β) at positions across the inserted phage rDNA in wild-type cell, ΔnusB cells, and ΔsuhB cells. Values indicate the background-subtracted fold enrichment above a control region in the transcriptionally silent bglB gene (see Materials and Methods). A schematic of the rRNA loci is drawn to scale and aligned with the data. Horizontal black lines indicate the position of the amplicons used for PCR quantification of the ChIP signal. boxA sequences are represented by red boxes. Data represent averages of three independent, biological replicates. Error bars indicate 1 standard deviation above and below the mean.

FIG 5

NusB association with elongating RNAP at rRNA loci is dependent upon SuhB. The level of NusB and RNAP (β) association across rRNA loci was measured using ChIP-qPCR for wild-type and ΔsuhB and ΔnusB strain cells. Data points indicate the level of NusB ChIP signal normalized to that of RNAP β. The position of each data point on the x axis indicates the center of the PCR amplicon. A schematic of the rRNA loci is drawn to scale, aligned with the data. boxA sequences are represented by red boxes. Data represent averages of three independent, biological replicates. Error bars indicate 1 standard deviation above and below the mean.