Chaitanya Jain

A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy.

A broadly applicable genetic strategy was developed for investigating RNA-protein interactions and applied to the HIV-1 Rev protein. By rapidly screening thousands of Rev-RNA interactions in Escherichia coli, we isolated Rev suppressor mutations that alleviated the deleterious effect of mutations in RRE stem-loop IIB, the high affinity RNA-binding site for Rev. All of these suppressor mutations map to a single arginine-deficient face of a Rev alpha-helix, and some alter the binding specificity of the protein, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev. The spatial constraints suggested by these data have enabled us to model the structure of this complex.

Structural model for the cooperative assembly of HIV-1 Rev multimers on the RRE as deduced from analysis of assembly-defective mutants

The functional efficacy of the HIV-1 Rev protein is highly dependent on its ability to assemble onto its HIV-1 RNA target (the RRE) as a multimeric complex. To elucidate the mechanism of multimeric assembly, we have devised two rapid and broadly applicable strategies for examining cooperative interactions between proteins bound to RNA, one based on cooperative translational repression of a two-site reporter and the other on gel shift analysis with crude E. coli extracts. Using these strategies, we have identified two distinct surfaces of Rev (head and tail) that are critical for different steps in multimeric assembly. Our data indicate that Rev assembles cooperatively on the RRE via a series of symmetrical tail-to-tail and head-to-head protein-protein interactions. The insights into molecular architecture suggested by these findings have enabled us to derive a structural model for Rev and its multimerization on the RRE.

Consequences of RNase E scarcity in Escherichia coli

The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate‐determining. Our data also indicate that E. coli growth requires a cellular RNase E concentration at least 10–20% of normal and that the feedback mecha‐nism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the in‐crease in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, when either mutated or cloned on a multicopy plasmid, seems to be capable of compensating for an inadequate supply of this essential protein.

RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling

The interaction of nucleolin with a short stem‐loop structure (NRE) requires two contiguous RNA‐binding domains (RBD 1+2). The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli. Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target. Mutations that alter RNA‐binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1+2 amino acids and nucleotides within the NRE stem‐loop. Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA. The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three‐dimensional model of nucleolin RBD 1+2 bound to the NRE stem‐loop.

IS 10 mRNA stability and steady state levels in Escherichia coli: indirect effects of translation and role of rne function

Translation of the IS 10 transposase gene is known to be very infrequent. We have identified mutations whose genetic properties suggest that they act directly to increase or decrease the intrinsic level of translation initiation. Also, we have analysed in detail the effects of these mutations on IS 10 mRNA using one particular IS 10 derivative. In this case, increases or decreases in translation are accompanied by increases or decreases in both the steady state level and the half‐life of transposase mRNA; effects on steady state levels are much more dramatic than effects on message half‐life. At wild‐type levels of translation initiation, the rate‐limiting step in physical decay of full length IS 10 message for a particular IS 10 derivative is shown to be rne‐dependent endonucleolytic cleavage; 3′ exonucleases appear to play a secondary role, degrading primary cleavage products. Analysis of interplay between translation mutations and rne function, together with the above observations, suggests that translation stabilizes messages in a general way against rne‐dependent endonucleolytic cleavage, and that significant protection may be conferred by one or a few ribosomes. However, dramatic effects of translation on steady state message levels are still observed in an rne mutant and involve the 3′ end of the transcript; we propose that these additional effects reflect translation‐mediated stimulation of transcript release.

Functional and molecular analysis of Escherichia coli strains lacking multiple DEAD-box helicases

DEAD-box RNA helicases are enzymes that unwind RNA duplexes and are found in virtually all organisms. Most organisms harbor multiple DEAD-box helicases, suggesting that these factors participate in distinct aspects of RNA metabolism. To define the individual and collective contribution of the five DEAD-box helicases in the bacterium Escherichia coli (E. coli), nonpolar deletion mutants lacking single or multiple DEAD-box genes were constructed. An analysis of the single-deletion strains indicated that the absence of either the DeaD or SrmB RNA helicase causes growth and/or ribosomal defects under typical laboratory growth conditions. The analysis of strains lacking multiple DEAD-box genes showed cumulative growth defects at low temperatures. A strain deleted for all five DEAD-box genes was also constructed for these studies, representing the first time all DEAD-box genes have been removed in any organism. Additional investigations revealed that the growth and ribosomal defects of such a DEAD-box deficient strain can be sharply attenuated under alternative conditions, indicating that the defects caused by a lack of DEAD-box genes are modulated by growth context.

RNase E Regulates the Yersinia Type 3 Secretion System

Yersinia spp. use a type 3 secretion system (T3SS) to directly inject six proteins into macrophages, and any impairment of this process results in a profound reduction in virulence. We previously showed that the exoribonuclease polynucleotide phosphorylase (PNPase) was required for optimal T3SS functioning in Yersinia pseudotuberculosis and Yersinia pestis. Here we report that Y. pseudotuberculosis cells with reduced RNase E activity are likewise impaired in T3SS functioning and that phenotypically they resemble Delta pnp cells. RNase E does not affect expression levels of the T3SS substrates but instead, like PNPase, regulates a terminal event in the secretion pathway. This similarity, together with the fact that RNase E and PNPase can be readily copurified from Y. pseudotuberculosis cell extracts, suggests that these two RNases regulate T3SS activity through a common mechanism. This is the first report that RNase E activity impacts the T3SS as well as playing a more general role in infectivity.

Preferential cis action of IS 10 transposase depends upon its mode of synthesis

A number of bacterial DNA‐binding proteins, including IS element transposases, act preferentially in cis. We show below that the degree of preferential cis action by IS 10 transposase depends upon its mode of synthesis at steps subsequent to transcription initiation. Cis preference is increased several fold by mutations that decrease translation initiation, by the presence of IS 10‐specific antisense RNA and by plasmids that increase the level of cellular RNases. Conversely, cis preference is decreased by mutations that increase translation initiation; in some cases, cis preference is nearly abolished. Mutations that alter the rate of transcription initiation have no effect. In light of other observations, we suggest that cis preference is strongly dependent upon the rate at which transcripts are released from their templates and/or the half‐life of the transposase message. These observations provide further evidence that inefficient translation plays multiple roles in the biology of IS 10.

Degradation of mRNA in Escherichia coli

Degradation of messenger RNAs (mRNAs) is a universal process that occurs in every cell and has important implications for nucleotide metabolism and gene expression. One organism in which mRNA degradation has been thoroughly studied is the bacterium Escherichia coli ( E. coli ). In this review I describe what is presently known about the different processes involved in the conversion of mRNAs from high molecular weight species to mononucleotides in E. coli . The ribonucleases and accessory factors involved in mRNA degradation, and features on mRNAs that make them resistant or sensitive to degradation will also be described. At the conclusion of this review, some of the anticipated directions of future research on this topic will be discussed.

New improved lacZ gene fusion vectors

New plasmid vectors suitable for creating fusions with the lacZ gene have been developed. These vectors represent an improvement over currently available vectors and possess the following features: (1) an undetectable background beta-galactosidase (beta Gal) activity in the absence of fusion, (2) an extended multiple cloning site (MCS), and (3) the ability to conveniently subclone in any one of three translational frames. Medium- and high-copy-number versions of these vectors have been developed.