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The majority of Escherichia coli mRNAs undergo post-transcriptional modification in exponentially growing cells.

Mohanty BK, Kushner SR - Nucleic Acids Res. (2006)

Bottom Line: Conversely, mRNAs terminated in a Rho-dependent fashion are probably not substrates for PAP I, but can be modified by the addition of long polynucleotide tails through the biosynthetic activity of polynucleotide phosphorylase (PNPase).Furthermore, real-time PCR analysis indicates that the extent of polyadenylation of individual full-length transcripts such as lpp and ompA varies significantly in wild-type cells.The data presented here demonstrates that polyadenylation in E.coli occurs much more frequently than previously envisioned.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Georgia, Athens, GA 30602, USA.

ABSTRACT
Polyadenylation of RNAs by poly(A) polymerase I (PAP I) in Escherichia coli plays a significant role in mRNA decay and general RNA quality control. However, many important features of this system, including the prevalence of polyadenylated mRNAs in the bacterium, are still poorly understood. By comparing the transcriptomes of wild-type and pcnB deletion strains using macroarray analysis, we demonstrate that >90% of E.coli open reading frames (ORFs) transcribed during exponential growth undergo some degree of polyadenylation by PAP I, either as full-length transcripts or decay intermediates. Detailed analysis of over 240 transcripts suggests that Rho-independent transcription terminators serve as polyadenylation signals. Conversely, mRNAs terminated in a Rho-dependent fashion are probably not substrates for PAP I, but can be modified by the addition of long polynucleotide tails through the biosynthetic activity of polynucleotide phosphorylase (PNPase). Furthermore, real-time PCR analysis indicates that the extent of polyadenylation of individual full-length transcripts such as lpp and ompA varies significantly in wild-type cells. The data presented here demonstrates that polyadenylation in E.coli occurs much more frequently than previously envisioned.

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Comparison of steady-state levels of representative mRNAs in wild-type (MG1693) and PAP I induced (SK9124) strains employing northern blot analysis. Cell cultures were grown to 50 Klett units (∼1 × 108cells/ml) above background (0 min). Subsequently IPTG (350 μM) was added and the cultures were grown for additional times. Total RNA was isolated at times (min after IPTG addition) indicated on the top of the blot. Five microgram of total RNA was loaded in each lane and separated in 6% polyacrylamide/7 M urea gels. The transcripts were probed as described in Materials and Methods. The relative quantity of each full-length transcript was determined using a Molecular Dynamics PhosphorImager. The wild-type level of each mRNA at 0 min was set at 1 and the corresponding fold-changes are noted at the bottom of each lane.
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fig2: Comparison of steady-state levels of representative mRNAs in wild-type (MG1693) and PAP I induced (SK9124) strains employing northern blot analysis. Cell cultures were grown to 50 Klett units (∼1 × 108cells/ml) above background (0 min). Subsequently IPTG (350 μM) was added and the cultures were grown for additional times. Total RNA was isolated at times (min after IPTG addition) indicated on the top of the blot. Five microgram of total RNA was loaded in each lane and separated in 6% polyacrylamide/7 M urea gels. The transcripts were probed as described in Materials and Methods. The relative quantity of each full-length transcript was determined using a Molecular Dynamics PhosphorImager. The wild-type level of each mRNA at 0 min was set at 1 and the corresponding fold-changes are noted at the bottom of each lane.

Mentions: However, these results seemed to contradict our expectations of reduced steady-state levels based on the shorter half-lives we observed after a 15 min induction (5). In particular, we previously reported that the half-lives of specific transcripts such as lpp, ompA, trxA, and rpsO were decreased ∼1.5- to 4-fold under conditions in which there was an ∼90-fold increase in the in vivo poly(A) level (5). As such, we carefully reexamined the half-life data and noticed that changes were not observed until 5–8 min after the addition of rifampicin (∼20–25 min of increased PAP I activity) (5). Accordingly, we hypothesized that the 15 min period over which PAP I was overproduced in the presence of ongoing transcription was too short to allow new steady-state levels to be established. To investigate this possibility directly, the steady-state mRNA levels of lpp, ompA, trxA and rpsO were determined for up to 45 min after PAP I induction. Under these circumstances, the growth rate of the PAP I induced strain decreased marginally from 30 min before induction to 35 min after induction (data not shown), while the steady-levels of all the transcripts tested decreased between 2.5- and 5.0-fold (Figure 2). These data suggest that more than 15 min of PAP I overexpression is required for transcripts to reach new steady-state levels.


The majority of Escherichia coli mRNAs undergo post-transcriptional modification in exponentially growing cells.

Mohanty BK, Kushner SR - Nucleic Acids Res. (2006)

Comparison of steady-state levels of representative mRNAs in wild-type (MG1693) and PAP I induced (SK9124) strains employing northern blot analysis. Cell cultures were grown to 50 Klett units (∼1 × 108cells/ml) above background (0 min). Subsequently IPTG (350 μM) was added and the cultures were grown for additional times. Total RNA was isolated at times (min after IPTG addition) indicated on the top of the blot. Five microgram of total RNA was loaded in each lane and separated in 6% polyacrylamide/7 M urea gels. The transcripts were probed as described in Materials and Methods. The relative quantity of each full-length transcript was determined using a Molecular Dynamics PhosphorImager. The wild-type level of each mRNA at 0 min was set at 1 and the corresponding fold-changes are noted at the bottom of each lane.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636475&req=5

fig2: Comparison of steady-state levels of representative mRNAs in wild-type (MG1693) and PAP I induced (SK9124) strains employing northern blot analysis. Cell cultures were grown to 50 Klett units (∼1 × 108cells/ml) above background (0 min). Subsequently IPTG (350 μM) was added and the cultures were grown for additional times. Total RNA was isolated at times (min after IPTG addition) indicated on the top of the blot. Five microgram of total RNA was loaded in each lane and separated in 6% polyacrylamide/7 M urea gels. The transcripts were probed as described in Materials and Methods. The relative quantity of each full-length transcript was determined using a Molecular Dynamics PhosphorImager. The wild-type level of each mRNA at 0 min was set at 1 and the corresponding fold-changes are noted at the bottom of each lane.
Mentions: However, these results seemed to contradict our expectations of reduced steady-state levels based on the shorter half-lives we observed after a 15 min induction (5). In particular, we previously reported that the half-lives of specific transcripts such as lpp, ompA, trxA, and rpsO were decreased ∼1.5- to 4-fold under conditions in which there was an ∼90-fold increase in the in vivo poly(A) level (5). As such, we carefully reexamined the half-life data and noticed that changes were not observed until 5–8 min after the addition of rifampicin (∼20–25 min of increased PAP I activity) (5). Accordingly, we hypothesized that the 15 min period over which PAP I was overproduced in the presence of ongoing transcription was too short to allow new steady-state levels to be established. To investigate this possibility directly, the steady-state mRNA levels of lpp, ompA, trxA and rpsO were determined for up to 45 min after PAP I induction. Under these circumstances, the growth rate of the PAP I induced strain decreased marginally from 30 min before induction to 35 min after induction (data not shown), while the steady-levels of all the transcripts tested decreased between 2.5- and 5.0-fold (Figure 2). These data suggest that more than 15 min of PAP I overexpression is required for transcripts to reach new steady-state levels.

Bottom Line: Conversely, mRNAs terminated in a Rho-dependent fashion are probably not substrates for PAP I, but can be modified by the addition of long polynucleotide tails through the biosynthetic activity of polynucleotide phosphorylase (PNPase).Furthermore, real-time PCR analysis indicates that the extent of polyadenylation of individual full-length transcripts such as lpp and ompA varies significantly in wild-type cells.The data presented here demonstrates that polyadenylation in E.coli occurs much more frequently than previously envisioned.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Georgia, Athens, GA 30602, USA.

ABSTRACT
Polyadenylation of RNAs by poly(A) polymerase I (PAP I) in Escherichia coli plays a significant role in mRNA decay and general RNA quality control. However, many important features of this system, including the prevalence of polyadenylated mRNAs in the bacterium, are still poorly understood. By comparing the transcriptomes of wild-type and pcnB deletion strains using macroarray analysis, we demonstrate that >90% of E.coli open reading frames (ORFs) transcribed during exponential growth undergo some degree of polyadenylation by PAP I, either as full-length transcripts or decay intermediates. Detailed analysis of over 240 transcripts suggests that Rho-independent transcription terminators serve as polyadenylation signals. Conversely, mRNAs terminated in a Rho-dependent fashion are probably not substrates for PAP I, but can be modified by the addition of long polynucleotide tails through the biosynthetic activity of polynucleotide phosphorylase (PNPase). Furthermore, real-time PCR analysis indicates that the extent of polyadenylation of individual full-length transcripts such as lpp and ompA varies significantly in wild-type cells. The data presented here demonstrates that polyadenylation in E.coli occurs much more frequently than previously envisioned.

Show MeSH
Related in: MedlinePlus