Research Interests:

In all biological systems, messenger RNAs (mRNAs) serve as intermediates in the conversion of the genetic information contained within a cell’s DNA into functional proteins.  In prokaryotes, such as Escherichia coli, mRNAs are both synthesized and degraded rapidly, providing the organism with an excellent mechanism for rapid adaptation to environmental changes.  The half-lives of mRNAs can vary greatly (20 sec-25 min in E. coli) but generally are not longer than the generation time of the bacterium.  A unique feature in bacteria is the existence of polycistronic mRNAs.  Many of these large transcripts are processed into smaller units as a means of regulating the expression of specific genes within each operon.  Another interesting aspect of prokaryotic mRNAs is the post-transcriptional addition of poly(A) tails.  Experiments from our laboratory (O'Hara et al.,  1995; Mohanty and Kushner, 1999a) and others have suggested that these tails serve as a targeting mechanism for rapid degradation. 

            In order to better understand this complex and important regulatory system, our laboratory has a number of projects underway that involve a combination of genetic, molecular biological, genomic, proteomic and biochemical approaches.  These include:

1. Analysis of  polyadenylation in Escherichia coli and other prokaryotes

The focus of this project is to understand the molecular mechanism of polyadenylation in E. coli. While it is clear that the addition of poly(A) tails leads to the more rapid decay of mRNAs (O'Hara et al., 1995), many questions remain, such as are there preferred polyadenylation sites, are there accessory proteins that assist poly(A) polymerase, does E. coli possess poly(A) binding proteins, and why is immature 23S ribosomal RNA a primary substrate for poly(A) polymerase (Mohanty and Kushner, 1999a).  In addition, the analysis of polyadenylation is complicated by the fact that while poly(A) polymerase accounts for 90% of the poly(A) tails (Mohanty and Kushner, 1999a), polynucleotide phosphorylase (PNPase) also synthesizes heteropolymeric tails in wild-type bacteria (Mohanty and Kushner, 2000b).  In fact, poly(A) polymerase and PNPase account for all the polyadenylation in exponentially growing E. coli (Mohanty and Kushner, 2000b).  Although polyadenylation is clearly a very complex system, we have made recent progress in understanding the interrelationship between poly(A) levels and the intracellular amounts of RNase E and PNPase (Mohanty and Kushner, 2002).   We are currently trying to determine whether polyadenylation is used primarily to target full-length mRNAs or to help promote the degradation of mRNA fragments generated by endonucleolytic digestion by enzymes such as RNase E,  RNase G and RNase III.  In addition, we are working to identify proteins other than poly(A) polymerases that are involved in the polyadenylation process.  A current working model of polyadenylation is shown in Fig. 1. 

Figure 1.  A current model for polyadenylation of mRNAs in E. coli.   mRNAs generated following Rho-independent transcription termination will contain a stem-loop structure at their 3' ends.  This structure inhibits the activity of both PNPase and RNase II.  In the presence of a potential accessory protein a complex containing PNPase and poly(A) polymerase I (PAP I) binds to the terminus.  In wild-type cells PAP I commences the addition of A residues to form a poly(A) tail.  At some point, PAP I is displaced by PNPase and degradation commences, proceeding through the stem-loop structure, releasing the accessory protein.  As PNPase proceeds towards the 5' end of the mRNA, its rate of degradation slows and eventually the enzyme begins to synthesize a heteropolymeric tail on the transcript.  A cycling process then ensues until the mRNA is either degraded into a small oligonucleotide (4-7 nt) that is no longer a substrate for PNPase or the enzyme dissociates from the substrate. 

2. Identification of the primary exonucleases involved in mRNA decay

In E. coli all of the riboexonucleases that have been identified degrade RNA molecules from the 3’ terminus.  These 3’ -> 5’ exonucleases fall into two classes depending on their mechanism of catalysis.  Hydrolytic enzymes such as RNase II and RNase R release nucleoside monophosphates, while enzymes such as polynucleotide phosphorylase and RNase PH use a phosphorolytic mechanism that requires the presence of inorganic phosphate and releases nucleoside diphosphates.  Polynucleotide phosphorylase is a reversible enzyme. Recently, we have shown that this enzyme functions both degradatively and biosynthetically in E. coli (Mohanty and Kushner, 2000b).  We are currently trying to determine which of the various ribonucleases are primarily involved in the degradation of mRNAs.  Genome-wide analysis using RNase II and PNPase deletion mutants has now demonstrated that PNPase plays a greater role in mRNA decay than RNase II (Mohanty and Kushner, 2003).  An unexpected observation from this work was the large percentage of E. coli mRNAs that are stabilized in the absence of RNase II (Mohanty and Kushner, 2003).  We are currently working to determine the molecular basis of this phenomenon. 

3. Analysis of the role of RNase E in mRNA decay

RNase E (Fig. 2) was first identified in the late 1970s based on its affect of the processing of a 9S rRNA precursor.  Subsequently, it was shown to be involved in mRNA decay and the maturation of tRNAs.  Since this enzyme is essential for cell viability, it was assumed that either the defect in mRNA decay or 9S rRNA processing was responsible for the loss of cell viability.  Experiments carried out with a series of RNase E deletion mutations demonstrated that these assumptions were not correct (Ow et al., 2000).  In fact, we have now shown that the essential function of RNase E in E. coli is its ability to initiate the maturation of tRNAs (Ow and Kushner, 2002).  Of further interest is the fact that RNase E serves as the scaffold for a multiprotein complex called the "degradosome" (Fig. 2).   Since this complex contains both endo- and exonucleases as well as an RNA helicase activity, it was assumed that it accounted for the bulk of mRNA decay.  However, analysis of RNase E deletion mutants has demonstrated that assembly of the degradosome is not required for normal mRNA decay (Ow et al., 2000).  In contrast, degradosome assembly is required for the cell to be able to detect alterations in the level of polyadenylation (Mohanty and Kushner, 2002a).  Additional experiments on RNase E have shown that the gene is controlled by a complex regulatory system that involves three distinct promoters (Ow et al., 2002).   Efforts are now underway to obtain the crystal structure of the catalytic region of RNase E (amino acids 1-500) and to determine the role of the arginine rich binding domain (AARBS) (Fig. 2).  In addition, we are employing a variety of genetic and biochemical techniques to help understand how RNase E distinguishes among mRNA, rRNA and tRNA substrates. 

Figure 2.  Comparison of RNase E and RNase G.  HSR 1 and HSR 2 are regions that  contain greater than 60% similarity between the two proteins.  ARRBS is an arginine-rich RNA binding site.  The degradosome scaffolding region serves as the platform for the association of polynucleotide phosphorylase (PNPase), the glycolytic enzyme enolase and the RhlB RNA helicase to form the degradosome.  S1 is a RNA binding domain first identified in the ribosomal protein S1. 

4. Analysis of the role of RNase G in mRNA decay

RNase G is a functional homologue of RNase E that is 34.1% identical over the first 488 amino acids of the RNase E protein (Fig. 2).  Many prokaryotes have homologues of at least one of these two proteins.  Although deletion of the structural gene for RNase G does not lead to any major phenotypic alterations, it has been shown that the protein is involved in the maturation of 16S rRNA and the decay of a few mRNAs.  By constructing a series of isogenic strains containing combinations of the rne-1 and rng::cat alleles, we have been able to demonstrate that RNase G can serve as a backup in both the decay of mRNAs and the processing of  a 9S rRNA precursor into the 5S rRNA (Ow et al., 2003).  However, RNase G is not able to process tRNA precursors (Ow et al., 2003).  We are currently working determining what distinguishes the two proteins in their ability to bind and cleave various RNA substrates by constructing chimeric proteins. 

5. Understanding the multiple pathways of mRNA decay

Over the past 15 years, we have constructed a large number of E. coli mutants that are deficient in one or more enzymes thought to be involved in mRNA decay.  In many of these strains we observed reduced rates of mRNA decay (Arriano et al., 1988; O'Hara et al., 1995; Granger et al., 1998).  However, we have not yet succeeded in constructing a mutant that is completely defective in mRNA decay.  Thus we continue to seek additional genes that are involved in the pathways of mRNA decay.  Our ultimate goal is to generate a comprehensive molecular map of mRNA decay in E. coli.   A current working model is described in Figure 3.

Figure 3.  Model for mRNA decay in Escherichia coli.  This model is based on data published in (Ow et al., 2003).  For simplicity sake, RNase E is not shown as part of the degradosome.  In A, mRNA decay is initiated by the binding of RNase E to the 5' terminus of the transcript, followed by cleavage at an internal site.  In B, a polycistronic transcript is cleaved in an intergenic region by RNase III.  For some transcripts (C) degradation does not involve any endonucleolytic cleavages but is carried out primarily by exonucleolytic attack by enzymes such as PNPase or RNase II.  RNase G does not bind efficiently to 5' termini that contain a triphosphate so it is hypothesized that it primarily cleaves degradation products that have been generated by either RNase E or RNase III.  Dotted lines indicate inefficient pathways. 5' triphosphates are shown in black while 5' monophosphates are shown in green.  Oligoribonuclease is necessary to degrade short oligoribonucleotides (4-7 nt) that are resistant to both PNPase and RNase II.