Exoribonuclease and endoribonuclease activities of RNase BN/RNase Z both function in vivo

Abstract

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.

FIGURE 1.

Loop region around Pro-l42 in E. coli RNase BN obstructs the RNA exit channel. Superimposed structures of E. coli RNase BN (Protein Data Bank entry 2CBN) and B. subtilis RNase Z (Protein Data Bank entry 2FK6) are shown in ribbon representations, colored as gray and pale green, respectively. Loop region 140–145 is highlighted in yellow for E. coli RNase BN and in red for B. subtilis RNase Z. The orientation shown looks into the RNase BN active site, with the putative exit channel at the back top of the figure. Residue Pro-142 in E. coli (EC) RNase BN is shown as sticks, and the position of the corresponding glycine in B. subtilis (BS) RNase Z is marked by a red sphere. The two zinc ions coordinated at the E. coli RNase BN active site are shown as blue spheres.

FIGURE 2.

Comparison of the activity of wild type and P142G mutant RNase BN on model RNA substrates. The reactions were carried out as described under “Experimental Procedures” with the addition of 1.7 μm purified wild type or mutant RNase BN and 10 μm 5′-³²P-labeled single-stranded A₁₇ or G₅A₁₂CCA-A₅ substrate in the presence of Mg²⁺. Portions were withdrawn at the indicated time points.

FIGURE 3.

Processing of tRNA precursors by wild type and mutant RNase BN. A, structure of the E. coli tRNA^SelC precursor with the 3′-terminal CCA sequence in boldface type. tRNA^SelC (0.05 μm) labeled with [³²P]pC at its 3′-end was treated with wild type or mutant (P142G) RNase BN (0.14 μm) in the presence of either Mg²⁺ at pH 7.5 or Co²⁺ at pH 6.5. Cleavage products were analyzed by 20% denaturing PAGE. The positions of the 5-nt endoribonucleolytic cleavage product (Endo-5′ nt) and the mononucleotide generated as a result of exoribonucleolytic trimming are indicated. B, the structure of the E. coli tRNA^PheV precursor is shown with the 3′-CCA sequence in boldface type. 3′-[³²P]pC-labeled tRNA^PheV was treated with wild type or mutant RNase BN and analyzed as described in A. The 7-nt endoribonucleolytic cleavage product (Endo-7′ nt) and the mononucleotide generated as a result of exoribonucleolytic trimming are indicated. Identification of the reaction products was determined with RNA oligonucleotides and with CMP as standards. The mononucleotide exonuclease product was also confirmed to be CMP by paper chromatography, as described (18, 19).

FIGURE 4.

Processing of phage tRNA^Pro-Ser precursor by RNase BN. The structure of phage T4 tRNA^Pro-Ser dimeric precursor is shown at the top, with the discriminator nucleotides in boldface type. Phage T4 tRNA^Pro-Ser precursor (12 nm), labeled at its 3′-end with [³²P]pC, as described under “Experimental Procedures,” was used as the substrate. Digestion with wild type and P142G mutant RNase BN (0.04 μm) was carried out in the presence of Mg²⁺ at pH 7.5. All bands shorter than the full-length tRNA^Pro-Ser precursor are incomplete transcripts that are labeled with [³²P]pCp. P, precursor RNA substrate.

FIGURE 5.

Northern analysis of tRNA^PheV. Total RNA samples were isolated from 10 ml of culture as described under “Experimental Procedures” and dissolved in gel loading buffer. Eight micrograms of RNA samples from each of strains MG1655 I⁻II⁻D⁻T⁻ and MG1655 I⁻II⁻D⁻T⁻ containing the P142G mutation in RNase BN and wild type MG1655(seq)rph⁺ were subjected to electrophoresis and Northern analysis as described under “Experimental Procedures.” Hybridization was at 37 °C overnight. M, mature RNA substrate.

FIGURE 6.

Effect of P142G mutation in RNase BN on cell growth in an exoribonuclease-deficient strain. Strains MG1655 I⁻II⁻D⁻T⁻PH⁻ and MG1655 I⁻II⁻D⁻T⁻PH⁻ containing the P142G mutation in RNase BN were grown in YT medium at 37 °C. Samples were withdrawn at various times to measure cell growth (A₆₀₀). The growth rate is plotted as the log of A₆₀₀ values as a function of time.

FIGURE 7.

Northern blot analysis of tRNA^Cys, tRNA^Tyr, and tRNA^Ala. RNA samples were prepared as described under “Experimental Procedures.” Eight micrograms of total RNA were subjected to electrophoresis and then transferred to a GeneScreen Plus membrane. Transfer RNAs were detected using a ³²P-labeled 5′-end-specific probe. M, mature RNA substrate.