Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA

Abstract

While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits [small ribosomal subunits (SSUs)] and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of Escherichia coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase-deficient form of this protein. Our data reveal cold sensitivity and altered ribosomal profiles are associated with a DeltaksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis-prompting discussion of KsgA as a possible antimicrobial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family.

Figure 1. Deletion of ksgA results in cold sensitivity and altered ribosome profiles

Growth characteristics and ribosome profiles of ΔksgA and its parental strain reveal cold sensitive ribosome biogenesis defects in the absence of ksgA. (A) Growth of ΔksgA and its parental strain were measured during growth at the specified temperature following inoculation of LB from saturated culture grown at 37° C. (B) Dilution plating experiments of saturated culture diluted in ten-fold increments on plain LB plates at 37° C, 25° C and 20° C. (C) Sucrose sedimentation profiles of ribosomes and ribosomal subunits were analyzed by monitering A₂₅₄ across a 10-40% gradient following centrifugation. Peaks corresponding to SSUs and 70S ribosomes are shaded in black and grey, respectively. Peak area was measured to determine the percent of free SSUs. (I) ΔksgA grown at 37° C, (II) parental strain grown at 37° C, (III) ΔksgA grown at 25° C, and (IV) parental strain grown at 25° C. Cells were grown to an OD₆₀₀ of ∼0.8.

Figure 2. Overexpression of KsgA and E66A KsgA alter growth characteristics of ΔksgA and alter the state of SSUs

Overexpression of KsgA rescues cold sensitive growth phenotypes while altering the distribution of free and ribosome containing SSUs while a catalytically inactive KsgA form drastically changes SSUs propensity to form ribosomes. (A) Dilution plating experiments of saturated culture diluted in ten-fold increments on LB plates while overexpressing empty vector, wild-type KsgA or E66A KsgA at 37° C and 25° C. (B) Fractionation of subunits and ribosomes as well as quantification of SSUs in the free form and those in 70S ribosomes were calculated as described for Figure 1. Percentages of free SSUs are represented below each pie chart corresponding to overexpression of empty vector, KsgA or E66A KsgA in (I) ΔksgA grown at 37° C, (II) parental strain grown at 37° C, (III) ΔksgA grown at 25° C, and (IV) parental strain grown at 25° C. For representative profiles see to Figure 4A and 4B.

Figure 3. KsgA impacts SSU rRNA processing in vivo

SSU rRNA processing was examined by agarose gel electrophoresis analysis. (A) Schematic of rRNA processing in E. coli (Li et al., 1999, Srivastava & Schlessinger, 1990). (B) Agarose gel electrophoresis analysis of total rRNA extracted from ΔksgA grown at 37° C (lane 1), parental strain grown at 37° C (lane 2), ΔksgA grown at 25° C (lane 3), and parental strain grown at 25° C (lane 4). The percent 17S of total SSU rRNA is presented (see experimental procedures). (C) Primer extension experiments using 16S rRNA extracted from SSU and SSU-like (see Figure 4) particles obtained from ΔksgA grown at 37° C overexpressing empty vector (lane 1) WT KsgA (lane 2) or E66A KsgA (lanes 3 and 4). (D) Agarose gel electrophoresis analysis performed on total rRNA from ΔksgA and parental strains grown at 37° C (lanes 1-3 and 4-6, respectively) and 25° C (lanes 7-9 and 10-12, respectively) with induction of empty, KsgA, or E66A KsgA vector. Experiments were performed as in (B) with 17S percentages presented below each lane.

Figure 4. KsgA and E66A KsgA associate with accumulated 30S ribosomal subunit peaks

Western detection of KsgA in sucrose gradient fractions of ΔksgA grown at 37° C was performed to show localization of (A) KsgA or (B) E66A KsgA within the gradient. Gradients were analyzed as in Figure 1 and an anti-S3 antibody was used to identify SSU containing particles. (C) Western analysis of ten-fold serial dilutions of cell lysate overexpressing WT or E66A KsgA (see Figure 4A and B) were probed with antibodies recognizing 6XHis and S3 to show relative expression levels. (D) Increasing concentrations of arabinose were used to vary expression of KsgA in ΔksgA cells grown at 37° C, and free SSUs were compared to the total SSU population when empty vector () or KsgA () is overexpressed. Cells were grown to an OD₆₀₀ of ∼0.5-0.6.

Figure 5. Model of proposed roles of KsgA in SSU Ribosome Biogenesis

Light blue figures represent the progression of SSU biogenesis in (A) the absence of KsgA, (B) presence of KsgA or (C) presence of catalytically inactive KsgA (E66A KsgA), from primary transcript to participation of the mature SSU in the translation cycle. Leader and trailer rRNA sequences are represented as black lines with final rRNA processing steps occurring concomitant with or following initiation of translation. This model illustrates the proposition that KsgA not only facilitates rRNA processing but also plays a regulatory role in the biogenesis cascade and the significance of methylation by KsgA in this function.