Sidney R. Kushner

Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli.

Using the polymerase chain reaction and standard recombinant DNA techniques, a series of new multipurpose low-copy-number (lcn) vectors, pWSK29, pWKS30, pWKS129 and pWKS130, have been constructed. Plasmids pWSK29 and pWKS30 carry the ampicillin-resistance marker (ApR), 20 unique cloning sites flanked by T7 and T3 RNA polymerase promoters, the lacZ alpha gene and the bacteriophage f1 origin of replication (ori) for production of single-stranded (ss) DNA in the presence of a helper phage. Plasmids pWSK129 and pWKS130 carry the kanamycin-resistance marker (KmR) and have 16 unique cloning sites flanked by T7 and T3 RNA polymerase promoters positioned within the lacZ alpha gene. Plasmids pWSK129 and pWKS130 also contain the f1 ori for the generation of ss DNA in the presence of a helper phage. All of the plasmids have an lcn of six to eight per cell. Each vector can be used for: (i) complementation analysis, (ii) generating unidirectional deletions with exonuclease III/S1 nuclease, (iii) DNA sequencing, (iv) high-level gene expression using T7 RNA polymerase, and (v) run-off transcription. They are very useful for analyzing genes encoding proteins which are toxic in Escherichia coli in high copy number.

mRNA Decay in Escherichia coli Comes of Age

When Apirion first proposed that mRNA decay in Escherichia coli involves a series of endo- and exonucleolytic events (2), the general working assumption was that the turnover of transcripts is a simple salvage pathway that is necessary for recycling of ribonucleotides. Although experimental data at that time indicated that mRNAs are rapidly degraded (11, 40) and that decay of individual transcripts is independent of length (9), the number and specificities of the enzymes that actually carry out transcript degradation were still open questions. Twenty-nine years and many experiments later, a much different picture has emerged. Not only is the pathway of mRNA decay far more complex than originally envisioned, but it apparently also plays an integral role in regulating the expression of many genes. While many important features of this system remain to be elucidated, this prospective attempts to convey the current state of knowledge. In addition, it focuses primarily on those areas where there are disagreements regarding important features of the mRNA decay process.

RNA Methylation under Heat Shock Control

Structural, biochemical, and genetic techniques were applied to investigate the function of FtsJ, a recently identified heat shock protein. FtsJ is well conserved, from bacteria to humans. The 1.5 A crystal structure of FtsJ in complex with its cofactor S-adenosylmethionine revealed that FtsJ has a methyltransferase fold. The molecular surface of FtsJ exposes a putative nucleic acid binding groove composed of highly conserved, positively charged residues. Substrate analysis showed that FtsJ methylates 23S rRNA within 50S ribosomal subunits in vitro and in vivo. Null mutations in ftsJ show a dramatically altered ribosome profile, a severe growth disadvantage, and a temperature-sensitive phenotype. Our results reveal an unexpected link between the heat shock response and RNA metabolism.

The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli

In Escherichia coli, the post‐transcriptional addition of poly(A) tails by poly(A) polymerase I (PAP I, pcnB) plays a significant role in cellular RNA metabolism. However, many important features of this system, including its regulation and the selection of polyadenylation sites, are still poorly understood. Here we show that the inactivation of Hfq (hfq), an abundant RNA‐binding protein, leads to the reduction in the ability of PAP I to add poly(A) tails at the 3′ termini of mRNAs containing Rho‐independent transcription terminators even though PAP I protein levels remain unchanged. Those poly(A) tails that are synthesized in the absence of Hfq are shorter in length, even in the absence of polynucleotide phosphorylase (PNPase), RNase II and RNase E. In fact, the biosynthetic activity of PNPase in the hfq single mutant is enhanced and it becomes the primary polynucleotide polymerase, adding heteropolymeric tails almost exclusively to 3′ truncated mRNAs. Surprisingly, both PNPase and Hfq co‐purified with His‐tagged PAP I under native conditions indicating a potential complex among these proteins. Immunoprecipitation experiments using PNPase‐ and Hfq‐specific antibodies confirmed the protein–protein interactions among PAP I, PNPase and Hfq. Analysis of mRNA half‐lives in hfq, ΔpcnB and hfq ΔpcnB mutants suggests that Hfq and PAP I function in the same mRNA decay pathway.

Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG‐inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half‐life of total pulse‐labelled RNA along with decreased half‐lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long‐term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3′‐terminus of 23S rRNA, a site that was not tailed in wild‐type cells. Quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) demonstrated an almost 20‐fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript‐dependent manner.

Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly.

We demonstrate here that the assembly of the RNase E‐based degradosome of Escherichia coli is not required for normal mRNA decay in vivo. In contrast, deletion of the arginine‐rich RNA binding site (ARRBS) from the RNase E protein slightly impairs mRNA decay. When both the degradosome scaffold region and the ARRBS are missing, mRNA decay is dramatically slowed, but 9S rRNA processing is almost normal. An extensive RNase E truncation mutation (rneδ610) had a more pronounced mRNA decay defect at 37°C than the temperature‐sensitive rne‐1 allele at 44°C. Taken together, these data suggest that the inviability associated with inactivation of RNase E is not related to defects in either mRNA decay or rRNA processing.

Genomic analysis in Escherichia coli demonstrates differential roles for polynucleotide phosphorylase and RNase II in mRNA abundance and decay

Previous work has shown that simultaneous inactivation of polynucleotide phosphorylase (PNPase) and RNase II (both 3′→ 5′ exonucleases) in Escherichia coli leads to the loss of cell viability and the accumulation of partially degraded mRNA species. In order to help to distinguish how these two enzymes globally affect the abundance and decay of mRNAs, we have carried out a genome‐wide analysis of the steady‐state levels of E. coli transcripts using deletion mutations in either rnb or pnp. The data show that, in exponentially growing cells, inactivation of PNPase leads to an increase in the steady‐state level of more expressed mRNAs (17.3%) than inactivation of RNase II (7.3%). In contrast, the steady‐state levels of a large number of E. coli mRNAs (31%) are decreased in the absence of RNase II, including almost all the ribosomal protein genes, suggesting that a major function of this enzyme is to protect specific mRNAs from the activity of other ribonucleases. Array data were confirmed by Northern analysis of 12 individual mRNAs. A comparison between the steady‐state levels and the half‐lives of individual mRNAs indicates that there may be a direct interaction between transcription and mRNA decay for some of the transcripts. In addition, results are presented to show significant phenotypic differences between the pnp‐7 point mutant and the pnpΔ683 deletion allele.

mRNA Decay in Prokaryotes and Eukaryotes: Different Approaches to a Similar Problem

Over the past 15 years considerable progress has been made in understanding the molecular mechanisms of mRNA decay in both prokaryotes and eukaryotes. Interestingly, unlike other important biological reactions such as DNA replication and repair, many features of mRNA decay differ between prokaryotes or eukaryotes. Even when a particular enzyme like poly(A) polymerase has been conserved, polyadenylation of mRNAs in prokaryotes appears to serve a very different function than it does in eukaryotes. Furthermore, while mRNA degrading multiprotein complexes have been identified in both prokaryotes and eukaryotes, their composition and biochemical mechanisms are significantly different. Accordingly, this review seeks to provide a concise comparison of our current knowledge regarding the pathways of mRNA decay in two model organisms, the prokaryote Escherichia coli and the eukaryote Saccharomyces cerevisiae. IUBMB Life, 56: 585‐594, 2004

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

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.

Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli

Poly(A) tails in Escherichia coli are hypothesized to provide unstructured single‐stranded substrates that facilitate the degradation of mRNAs by ribonucleases. Here, we have investigated the role that such nucleases play in modulating polyadenylation in vivo by measuring total poly(A) levels, polyadenylation of specific transcripts, growth rates and cell viabilities in strains containing various amounts of poly(A) polymerase I (PAP I), polynucleotide phosphorylase (PNPase), RNase II and RNase E. The results demonstrate that both PNPase and RNase II are directly involved in regulating total in vivo poly(A) levels. RNase II is primarily responsible for degrading poly(A) tails associated with 23S rRNA, whereas PNPase is more effective in modulating the polyadenylation of the lpp and 16S rRNA transcripts. In contrast, RNase E appears to affect poly(A) levels indirectly through the generation of new 3′ termini that serve as substrates for PAP I. In addition, whereas excess PNPase suppresses polyadenylation by more than 70%, the toxicity associated with increased poly(A) levels is not reduced. Conversely, toxicity is significantly reduced in the presence of excess RNase II. Overproduction of RNase E leads to increased polyadenylation and no reduction in toxicity.