An mRNA degrading complex in Rhodobacter capsulatus

Abstract

An RNA degrading, high molecular weight complex was purified from Rhodobacter capsulatus. N-terminal sequencing, glycerol-gradient centrifugation, and immunoaffinity purification as well as functional assays were used to determine the physical and biochemical characteristics of the complex. The complex comprises RNase E and two DEAD-box RNA helicases of 74 and 65 kDa, respectively. Most surprisingly, the transcription termination factor Rho is a major, firmly associated component of the degradosome.

Figure 1

A model of the bacterial degradosome. This scheme presents current knowledge of the structural organization of the degradosome and its mode of action. NDPs inhibit PNPase, poly-phosphate probably inhibits the helicase. The model also depicts the current ideas about the interaction of known degradosome components. The ATP-dependent helicase dissolves RNA secondary structure and makes the RNA accessible for PNPase. PPK recycles ATP from NDPs; the role of enolase is still elusive. Ortho-phosphate P_i, poly-phosphate (P_i)_n, dinucleotides NDP. + or – indicate stimulatory or inhibitory influence on mRNA degradation. (PPK, poly-phosphate-kinase; PNPase, polynucleotide-phosphorylase). The figure is adapted from Rauhut and Klug (1).

Figure 2

(A) Purification of the R.capsulatus degradosome. The figure shows the protein pattern observed during different steps of the purification (left gel 3.5–14.5% PAA, right gel 8% PAA). Lane 1, flow-through from SP column; lanes 2 and 3, salt washes of same column; lane 4, eluted proteins from this column (1 M salt); lane 5, degradosome proteins as observed in fraction 8 of the glycerol gradient; lane 6, molecular weight standard (kDa). (B) Degradation assay. The diagram shows the pZBP substrate in the puf context. RNase E cleavage at site E₁ initiates in vivo degradation of the 2.7 kb pufBALMX mRNA. In vitro, both RNase E sites, E₁ and E₂, are processed. Pins indicate RNA secondary structure. (LHI, light harvesting complex I proteins; RC, reaction center proteins; t_1/2, observed half-lives for the 2.7 and the pufBA 0.5 kb messenger.) For a typical activity test, shown in the gel picture, 5000 c.p.m. of [α-³²P]UTP labeled pZBP RNA were incubated with 2 µl of diluted gradient fraction 9 at 30°C for the time indicated. Fragments were separated on a 7 M urea PAA-gel. ST, RNA calibration standard, numbers indicating nucleotides.

Figure 3

Glycerol-gradient centrifugation of the R.capsulatus degradosome. (A) The collected fractions from fraction 2 (top) to 16 (bottom). Numbers indicating molecular masses in kDa. ST, protein standards. (B) The sedimentation of gradient calibration proteins β-galactosidase (116 kDa subunit; top) and catalase (240 kDa; bottom). (C) The PNPase assay. In all three panels identical fractions are aligned vertically. Protein size indicated in kDa.

Figure 4

(A) Immunodetection of PNPase with E.coli PNPase antibody. Lane 1, E.coli extract; lanes 2 and 3, R.capsulatus glycerol gradient, fractions 8 and 10, respectively; lane 4, R.capsulatus SP fraction; M, protein marker (kDa). (B) The gel shows the proteins eluted from an anti-RNase E immunoaffinity resin. Lane 1, molecular weight standard (kDa); lane 2, precipitation with RNase added; lane 3, without RNase; lane 4, fractions loaded. Band of antibody heavy chain (HC).

Figure 5

Helicase assay. Degradation of the 318 nt substrate is monitored during a 30 min incubation in the presence or absence of ATP. Intermediates are shown to the left. Zero minute samples were prepared in the presence and control C in the absence of added protein. M, RNA length standard (nt). The gel on the left shows the degradation pattern when substrate is incubated with PNPase only.