(Received for publication, August 25, 1995)
From the
Ribonuclease II (RNase II) is a major exonuclease in Escherichia coli that hydrolyzes single-stranded
polyribonucleotides processively in the 3` to 5` direction. To
understand the role of RNase II in the decay of messenger RNA, a strain
overexpressing the rnb gene was constructed. Induction
resulted in a 300-fold increase in RNase II activity in crude extracts
prepared from the overexpressing strain compared to that of a
non-overexpressing strain. The recombinant polypeptide (Rnb) was
purified to apparent homogeneity in a rapid, simple procedure using
conventional chromatographic techniques and/or fast protein liquid
chromatography to a final specific activity of 4,100 units/mg.
Additionally, a truncated Rnb polypeptide was purified, solubilized,
and successfully renatured from inclusion bodies. The recombinant Rnb
polypeptide was active against both [H]poly(A) as
well as a novel (synthetic partial duplex) RNA substrate. The data show
that the Rnb polypeptide can disengage from its substrate upon stalling
at a region of secondary structure and reassociate with a new free
3`-end. The stalled substrate formed by the dissociation event cannot
compete for the Rnb polypeptide, demonstrating that duplexed RNAs
lacking 10 protruding unpaired nucleotides are not substrates for RNase
II. In addition, RNA that has been previously trimmed back to a region
of secondary structure with purified Rnb polypeptide is not a substrate
for polynucleotide phosphorylase-like activity in crude extracts. The
implications for mRNA degradation and the proposed role for RNase II as
a repressor of degradation are discussed.
Because the rate of synthesis of any given protein is directly
proportional to the concentration of its message, regulating the
balance between mRNA decay and its synthesis is an important aspect of
gene expression. In Escherichia coli, it is widely accepted
that mRNA decay is initiated by a series of endonucleolytic cleavages
catalyzed by RNase E (1, 2, 3) or
occasionally by RNase III (4, 5) followed by
processive exonucleolytic degradation of the message to oligo- and
mononucleotides(1, 2, 3) . Two
3`-exonucleases have been implicated in this process: ribonuclease II
(RNase II) ()and polynucleotide phosphorylase
(PNPase)(6) . RNase II, which is responsible for the majority
of the exonucleolytic activity in E. coli extracts (7) , hydrolyzes RNA to release 5`-mononucleotides(8) ,
while PNPase phosphorylyzes RNA to mononucleoside
diphosphates(9) . Although RNase II activity was first
described over three decades ago (10, 11) and purified
from whole cells several years
later(12, 13, 14) , details of its role in
mRNA degradation are still poorly understood.
RNA structure, known to be an important determinant of mRNA stability, can protect upstream sequences from digestion by the 3`-exonucleases. The Rho-independent terminator sequence (trp t) of the tryptophan operon (15) and the intergenic (malE-malF) REP sequence of the maltose operon (16) are classic examples of secondary structures that protect upstream RNA from 3`-exonucleolytic degradation both in vitro and in vivo. These investigations implied that the observed protection by 3`-stem-loop structures was the result of an impediment to the processive activities of RNase II and, to a lesser extent, of PNPase (15, 16, 17) . Recent observations of the decay of RNA-OUT, the antisense RNA that regulates Tn10/IS10 transposition, demonstrate that the higher the thermal stability of the RNA structure, the larger the barrier to degradation by RNase II(18) . Degradation by PNPase is much less affected by the relative stability of the RNA-OUT structure(18) . Interestingly, RNA-OUT appears to be stabilized approximately 3-fold against PNPase attack by RNase II(18) . In addition, the rpsO mRNA is also stabilized significantly by the presence of RNase II(19) . Although the mechanism by which RNase II shelters upstream sequences from further exonucleolytic attack is not understood, the observed protection was attributed to the formation of a stable RNase II-RNA complex, which sequesters the 3`-end of the transcript(18, 19) .
As part of the investigation of the functional and biophysical properties of RNase II and its role in the overall decay of mRNA, we have overexpressed RNase II and developed a rapid and simple purification of the enzyme free of other nucleases. The purified enzyme was used to investigate the mechanisms by which stem-loop structures impede exonucleases and the ability of RNase II to act as a repressor of PNPase activity.
Figure 1: 3`-Exonucleolytic degradation of a partial duplex RNA substrate. Panel a presents a schematic diagram of the 3`-exonucleolytic degradation of the 92-nucleotide RNA substrate on the left to the 77-nucleotide product on the right. A time course digestion of the synthetic RNA transcript t40B (a) as described under ``Experimental Procedures'' is shown in panels b and c. Crude extracts (S-150 fraction) prepared from strain CF881 (b) or strain 18-11 (c) were added to a final concentration of 0.5 µg/ml and incubated with substrate at 37 °C. Aliquots were removed from the reaction mixture at the times indicated (in minutes) as described under ``Experimental Procedures.'' The digestion products were analyzed by electrophoresis through a 10% polyacrylamide gel under denaturing conditions. NP denotes a control lane containing substrate incubated in the absence of protein for 60 min. The 92-nucleotide substrate (S) and the stable degradative intermediate (P) (77 nucleotides) are indicated with arrows.
Figure 2: PNPase is unable to attack partially digested t40B. a, crude extracts (S-30 fraction) prepared from strain 18-11 at a final concentration of 0.2 mg/ml were incubated with t40B in the absence (lanes 1-4) or presence (lanes 5-8) of 10 mM phosphate at 37 °C. b, extracts (S-30 fraction) prepared from strain 18-11 at a final concentration of 0.2 mg/ml were incubated in the presence of 10 mM phosphate at 37 °C with t40B, which had been previously digested to 77 nucleotides with purified Rnb polypeptide, extracted with phenol/chloroform, and precipitated with ethanol. Aliquots were removed from the reaction mixture at the times indicated (in minutes) as described under ``Experimental Procedures.'' The digestion products were analyzed by electrophoresis through a 10% polyacrylamide gel under denaturing conditions. The 92-nucleotide substrate (S), previously digested substrate (S*), and the 77-nucleotide degradative intermediate (P) are indicated with arrows. NP denotes a control lane containing substrate incubated in the absence of protein for 30 min.
Figure 3: Strategy for cloning and overexpression of rnb. Panel a illustrates a linear representation of the rnb gene of E. coli and its flanking sequences. Plasmids pGC100 (b) and pGC101 (c) were constructed by polymerase chain reaction as described in the text. The solid box represents the cloned sequence, and the line represents sequences derived from the vector pET-11. Coordinates of the rnb sequence and the predicted start and stop codons are shown in a. The Shine-Dalgarno sequence is represented by an enclosed SD, and B denotes the position of the BamHI restriction sites. The open box represents regions 3` to the rnb gene also cloned into plasmid pGC100 (b). Plasmid pGC101 (c) lacks 29 nucleotide residues deleted from the 3`-end of the rnb coding sequence. This deletion has extended the open reading frame 66 nucleotide residues into the T7 terminator region of pET-11 shown by the hatched box. The ``TGA'' stop codon at position 2078, predicted by the original rnb sequence(23) , is not in frame in the corrected sequence.
Upon induction
of cultures of GC100 or GC101 with IPTG, the Rnb polypeptide was
expressed to the extent that it represented the most abundant
polypeptide in whole cell extracts and a significant fraction of the
total cellular protein (Fig. 4, lane 3). When assayed
against poly(A), crude extracts (S-30) from strain GC100 displayed a
specific activity of 1,184 units/mg (Table 1), 300-fold higher
than that obtained from crude extracts prepared from the haploid strain
CF881 (specific activity = 3.9 units/mg). An efficient method of
purification was developed in part by exploiting several effective
steps from previously published
methods(12, 13, 14) . The initial step relies
on Cibacron blue-agarose chromatography to remove the bulk of the
nucleic acids and contaminating proteins while the majority (>90%)
of the Rnb polypeptide remains bound to the column. Considerable
efficiency was gained by loading the 3 M NaCl eluate from this
column directly onto a hydroxylapatite column. This proved to be an
invaluable step in the purification method since concentration,
desalting, and significant purification of the Rnb polypeptide could
take place in a single step. The apparent loss of activity after
hydroxylapatite chromatography (Table 1) may have been due to the
inhibition by Ca ions leached from the column at high
ionic strength, as Ca
has been reported to inhibit
RNase II activity(10, 11) . Final purification of Rnb
from most contaminants could be achieved by affinity chromatography on
heparin-agarose or by ion exchange chromatography (FPLC). A sample of
the purified Rnb polypeptide is shown in Fig. 4, lanes 7 and 8. Based upon Coomassie Blue or silver staining of
overloaded polyacrylamide gels, the preparation was judged to be about
95% pure with a few faint minor contaminating bands. The specific
activity of the Rnb polypeptide purified to the end of the
heparin-agarose step was determined to be 4,100 units/mg, which is
nearly 2-fold greater than that reported by others for the enzyme
purified from whole cells(11, 12, 13) .
However, the specific activity of this preparation is approximately
2.5-fold lower than the best reported purification(14) . It is
quite possible that not all of the overexpressed Rnb polypeptide is
properly folded or fully active. Nonetheless, this method provides a
more rapid and facile purification of RNase II with good yields and
activity.
Figure 4: Purity of the recombinant Rnb polypeptide. Cultures of GC100 were induced with IPTG and grown for 5 h at 30 °C prior to harvest, lysis, and purification as described under ``Experimental Procedures.'' The following samples were denatured, separated in a 13% SDS-polyacrylamide gel, and stained with Coomassie Blue: lane 1, molecular mass standards (Bio-Rad); lane 2, boiled cell extract from a noninduced culture of GC100; lane 3, boiled cell extract from an induced culture of GC100; lane 4, S-30 extract (10 µg); lane 5, pooled fractions obtained from chromatography of the S-30 fraction on blue-agarose (BA) (2.5 µg); lane 6, pooled fractions from chromatography of the BA fraction on hydroxylapatite (HTP) (1.75 µg); lane 7, pooled fractions from chromatography of the HTP fraction on Resource Q (Q) (1.5 µg); and lane 8, pooled fractions from chromatography of the HTP fraction on heparin-agarose (HA) (1.5 µg).
If recoveries from the heparin-agarose chromatography step in Table 1are extrapolated to include all the material in pool A from the hydroxylapatite column, the overall yield is 29%. This apparent overall yield is low for two reasons. First, the activity in crude extracts represents the sum of activities of a number of endo- and exonucleases and overstates the activity of RNase II. Second, fractions were pooled to maximize purity rather than yield particularly after hydroxylapatite chromatography.
The addition of 22 amino acid residues, derived from the vector pET-11, to the C terminus of the truncated Rnb* polypeptide, resulted in the formation of insoluble inclusion bodies upon induction of cultures of GC101 with IPTG. The inclusion bodies were subsequently purified to near homogeneity by differential centrifugation in the presence of detergent. Authentic RNase II activity was recovered following solubilization, reduction, and refolding of the truncated Rnb* polypeptide from the inclusion bodies. Further purification of the renatured truncated Rnb* polypeptide from most contaminants could be achieved by ion exchange chromatography (FPLC). The truncated Rnb* polypeptide eluted from the Resource Q column over a broad range of NaCl concentrations likely reflecting the several different populations of misfolded and inactive polypeptides present in the preparation. RNase II activity eluted from the column as a sharp peak at a NaCl concentration of 220 mM. Although a significant amount of activity could be recovered from the inclusion bodies, the specific activity of this preparation was quite poor, 54 milliunits/mg, a small fraction of that obtained for the full-length Rnb polypeptide.
Figure 5: a, degradation of the partial duplex RNA substrate by purified Rnb polypeptide. In lanes 2-6, the t40B transcript was incubated with the purified Rnb polypeptide (1.2 milliunits; 5 ng/ml) for the indicated times (in minutes) at 37 °C as described under ``Experimental Procedures.'' Lanes 7 and 8 show a 60-min digestion of the t40B transcript incubated with the purified Rnb polypeptide (2.0 milliunits and 4.1 units; 8.3 ng/ml and 16 µg/ml, respectively). The digestion products were subsequently analyzed by gel electrophoresis. b, thermal inactivation of the recombinant Rnb polypeptide. Rnb polypeptide (0.5 µg/ml) was incubated in the absence of substrate for the indicated times (in minutes) prior to addition to a complete reaction mixture. Incubation was continued for an additional 15 min at a final concentration of Rnb polypeptide of 8.3 ng/ml. The digestion products were analyzed by gel electrophoresis. The substrate (S) and the degradative intermediate (P) are indicated by arrows, while the shorter degradation product shown in lane 8 is indicated by an arrowhead. NP denotes control lanes containing substrate incubated in the absence of protein for 60 min.
Digestion of the t40B transcript is complete after a 60-min incubation with 2.0 milliunits of RNase II activity (Fig. 5a, lane 7). Approximately 20% of the substrate is resistant to degradation by the Rnb polypeptide even after addition of 200 milliunits of fresh enzyme (data not shown). A fraction of the substrate appears to form concatemers and as a result does not have free 3`-ends accessible to the enzyme. Interestingly, digestion of t40B for 60 min at 37 °C with 4 units of enzyme resulted in a further shortened (73 nt) but stable degradation intermediate depicted by the arrowhead (Fig. 5a, lane 8). This experiment suggests that at high concentrations, the Rnb polypeptide can remove three to four additional unpaired residues in the t40B duplex remaining from a previous round of digestion.
Several previously published
reports have suggested that RNase II, of varying degrees of purity, is
readily inactivated by
heat(10, 11, 12, 13, 28) .
We have tested the purified Rnb polypeptide and found that the
recombinant enzyme is also susceptible to thermal inactivation (Fig. 5b). A comparison of Fig. 5b, lanes 2 and 3, shows that less than 1% of the
activity remains after a 5-min incubation of the Rnb polypeptide in the
absence of substrate at 37 °C. Interestingly, the enzyme is
stabilized in the presence of substrate and can remain active up to 60
min at 37 °C (Fig. 5a). Activity can also be
stabilized by the addition of substrate to Rnb polypeptide, which has
been partially inactivated by a brief incubation in buffer at 37
°C. Once activity has been lost to thermal inactivation, however,
it cannot be regained upon addition of substrate (data not shown). The
77-nt product also stabilized the enzyme against heating. Rnb
polypeptide was incubated in the presence of 1.5 pmol of partially
digested t40B for 5 min at 37 °C prior to incubation with
full-length t40B transcript. The 77-nt product not only protected the
Rnb polypeptide against thermal inactivation but also appeared to
stimulate the activity of the enzyme for the full-length substrate by
approximately 2-fold (data not shown). The apparent stimulation may be
attributable to a decreased rate of thermal inactivation. Taken
together, the data demonstrate that the enzyme can be stabilized by
both substrate and product. In contrast, both a single-stranded DNA
oligonucleotide (33-mer) and double-stranded plasmid DNA inhibited the
activity of RNase II but were unable to provide significant protection
from heating (data not shown) unlike oligonucleotides of
deoxy(C), which can reduce the rate of thermal
inactivation(28) .
Stabilization of RNase II by the digested t40B transcript implies that in the absence of any free 3`-single-stranded ends, the Rnb polypeptide can bind RNA even if it is not a substrate. To test this hypothesis, t40B was incubated briefly with a large excess of Rnb polypeptide, sufficient to digest it to 73 nt, and then subjected to UV photocross-linking. Fig. 6, lane 1, shows labeling of a band of 70 kDa, the size expected for the Rnb polypeptide. In addition, there is label associated with a band of 14 kDa, which we believe to be RNase A. The Rnb polypeptide is, therefore, able to bind its product (Fig. 6, lane 1) in the absence of any other proteins or cofactors. A 70-kDa protein, corresponding to the molecular mass of RNase II, was also labeled in crude extracts prepared from strain CF881 (Fig. 6, lane 3). All bands were sensitive to proteinase K treatment (Fig. 6, lanes 2 and 4). A comparison of Fig. 6, lanes 1 and 3, also demonstrates that UV cross-linking can provide an important assessment of the purity of the enzyme preparation in light of the affinity chromatography techniques utilized in the purification. Since there are a large number of RNA binding proteins in crude extracts prepared from E. coli that have a significant affinity for the t40B transcript, the presence of even a small percentage these contaminants would be readily detected in the purified material (Fig. 6, compare lane 3 to lane 1).
Figure 6:
UV cross-linking of t40B to purified
recombinant Rnb polypeptide and proteins in the S-150 fraction prepared
from strain CF881. Labeled t40B was incubated with purified recombinant
Rnb polypeptide (2.1 units, 50 µg/ml) or with 10 µg of an S-150
fraction prepared from strain CF881 (33 milliunits) at a concentration
of 1 mg/ml, irradiated with UV, digested with ribonucleases, and then
separated by SDS-PAGE as described under ``Experimental
Procedures.'' A duplicate sample was treated with proteinase
K() (PROT K) prior to electrophoresis (lanes 2 and 4). Lanes 1 and 2, purified
recombinant Rnb polypeptide; lanes 3 and 4, crude
extract prepared from strain CF881.
We have also tested whether the 77-nt product would inhibit the activity of the Rnb polypeptide in subsequent rounds of digestion. In the first experiment, the Rnb polypeptide (3.3 milliunits) was incubated with 25 pmol of unlabeled t40B (2.5-fold molar excess over labeled t40B) for 2.5 min at 37 °C prior to addition of labeled t40B. The kinetics of digestion of labeled t40B over a 60-min time course were identical to those in an incubation in which the same amount of enzyme was incubated directly with labeled t40B (data not shown). In the second experiment, the t40B transcript, which had been previously digested with Rnb polypeptide, extracted with phenol/chloroform, and ethanol precipitated, was used in a competition experiment. Equimolar amounts of digested t40B did not alter the kinetics of disappearance of the 92-nt substrate and thus were unable to compete effectively for the Rnb polypeptide (Fig. 7). Although the 77-nt product can protect the enzyme from thermal inactivation, it cannot inhibit its activity.
Figure 7:
Competition between partially digested
t40B and complete t40B. Rnb polypeptide (2.0 milliunits, 8.3 ng/ml) was
incubated with intact t40B in the presence () or absence
(
) of 10 pmol of t40B that had been previously digested to yield
a 77-nucleotide product with the purified Rnb polypeptide. The products
were resolved by gel electrophoresis, and the relative amounts of t40B
were quantified with a PhosphorImager, expressed as picomoles of RNA
remaining, and plotted as a function of
time.
Recent observations have suggested that RNase II can protect ``upstream'' RNA sequences from PNPase attack through the formation of a stable RNA-RNase II complex(18, 19) . We have further investigated this hypothesis by incubating the 77-nt product, produced by the action of the Rnb polypeptide (see above), with crude extracts prepared from strain 18-11 in the presence of 10 mM sodium phosphate. The data demonstrate that the 77-nt product is resistant to digestion by a PNPase-like activity (Fig. 2b, lanes 1-4). As discussed above, the 92-nt substrate is rapidly shortened to approximately 77 nt in the presence of phosphate over a 30-min time course of digestion (Fig. 2a, lanes 5-8).
The linear kinetics observed for the reaction demonstrate that RNase II stalls at regions of secondary structure, however briefly, but can disengage from the ``stalled'' substrate and reassociate with a new free 3`-end. This is substantiated by the demonstration that the Rnb polypeptide can cycle from an unlabeled to a labeled substrate. Our finding of dissociation from a substrate with 9 unpaired protruding nucleotides at the 3`-end of the 77-nt product is in good agreement with the 10-15-nt digestion limit product obtained for RNase II acting on homopolymers(28) . Interestingly, RNase II can participate in the processing of some tRNAs in vitro by degrading long trailing sequences but must be able to dissociate from the precursor to allow final maturation of the tRNA by other processing exonucleases(29) .
Two lines of evidence suggest that the Rnb polypeptide can also reassociate with the 77-nt product of digestion. First, at high concentrations of enzyme the Rnb polypeptide can remove three to four additional unpaired residues remaining from a previous round of digestion. Second, the Rnb polypeptide can bind its 73-77-nt product as evidenced by UV cross-linking and protection from thermal inactivation. Although partially digested t40B can bind to the Rnb polypeptide, it does not compete with the full-length substrate, indicating that the preferred substrate for RNase II has an extended free 3`-end. Moreover, the lack of competition by product implies that product binds to a site distinct from that of the substrate.
These observations suggest a possible model for the control of mRNA degradation at the 3`-end. As RNase II encounters a region of secondary structure, it stalls. If the structure is unstable, the enzyme may advance through the stem-loop in the 3` to 5` direction. However, if the structure is a stable REP sequence or a Rho-independent terminator, RNase II will dissociate from the transcript before the duplex opens. We propose that loss of the single-stranded 3` overhang, which reduces the affinity of RNase II for the stalled transcript, may also reduce the ability of the much larger PNPase to bind and degrade transcripts. It could also reduce the affinity of putative RNA helicases for such structures. Addition of a new 3`-end by PcnB followed by the action of PNPase, known to be less susceptible to RNA secondary structure than RNase II(15, 16, 17, 18, 19) , would be required for the degradation of strong REP and terminator sequences. Thus, a competition between removal of a 3`-overhanging sequence by RNase II and extension-degradation by PcnB and PNPase, respectively, would develop at the 3`-end of extended RNA secondary structures and may account for the heterogeneity in the 3`-ends of oligoadenylated RNA I(33) .
Note Added
in Proof-We have discovered that an A G transition,
resulting in a single amino acid change, Ser
Gly
, was inadvertantly incorporated into plasmic pGC100.