Real time analysis of the RNAI-RNAII-Rop complex by surface plasmon resonance: from a decaying surface to a standard kinetic analysis

RNA loop-loop complexes are motifs that regulate biological functions in both prokaryotic and eukaryotic organisms. In E. coli, RNAI, an antisense RNA encoded by the ColE1 plasmid, regulates the plasmid replication by recognizing through loop-loop interactions RNAII, the RNA primer that binds to the plasmidic DNA to initiate the replication. Rop, a plasmid-encoded homodimeric protein interacts with this transient RNAI-RNAII kissing complex. A surface plasmon resonance (SPR)-based biosensor was used to investigate this protein-nucleic acid ternary complex, at 5 degrees C, in experimental conditions such as the protein binds either to the loop-loop complex while it dissociates or to a saturated stable RNAI-RNAII surface. The results show that RNAI hairpin dissociates from the RNAII surface 110 times slower in the presence of Rop than in its absence. Rop binds to RNAI-RNAII with an on-rate of 3.6 x 10(6) M(-1) s(-1) and an off-rate of 0.11 s(-1), resulting in a binding equilibrium constant equal to 31 nM. A Scatchard-plot analysis of the interaction monitored by SPR confirms a 1:1 complex of Rop and RNAI-RNAII as observed for non-natural Rop-loop-loop complexes.     

Polyadenylation can regulate ColE1 type plasmid copy number independently of any effect on RNAI decay by decreasing the interaction of antisense RNAI with its RNAII target

Replication of ColE1-type plasmids is regulated by RNAI, an antisense RNA that interacts with the replication pre-primer, RNAII. Exonucleolytic attack at the 3' end of RNAI is impeded in pcnB mutant bacteria, which lack poly(A) polymerase I-the principal RNA polyadenylase of E. coli; this leads to accumulation of an RNAI decay intermediate (RNAI(-5)) and dramatic reduction of the plasmid copy number. Here, we report that polyadenylation can also affect RNAI-mediated control of plasmid DNA replication by inhibiting interaction of RNAI(-5) with RNAII. We show that mutation of the host pcnB gene profoundly affects the plasmid copy number, even under experimental conditions that limit the effects of polyadenylation on RNAI(-5) decay. Moreover, poly(A) tails interfere with RNAI/RNAII interaction in vitro without producing any detectable alteration of RNAI secondary structure. Our results establish the existence of a previously undetected mechanism by which RNA polyadenylation can control plasmid copy number.     

PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of CoIE1-related plasmids

The replication of CoIE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for pcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5′-end processing by RNAse E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST- PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.     

Suppression of RNA interference by adenovirus virus-associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.     

An Experimental Tool to Estimate the Probability of a Nucleotide Presence in the Crystal Structures of the Nucleotide–Protein Complexes

A correlation between the ligand–protein affinity and the identification of the ligand in the experimental electron density maps obtained by X-ray crystallography has been tested for a number of RNA-binding proteins. Bacterial translation regulators ProQ, TRAP, Rop, and Hfq together with their archaeal homologues SmAP have been used. The equilibrium dissociation constants for the N-methyl-anthraniloyl-labelled adenosine and guanosine monophosphates titrated by the proteins have been determined by the fluorescent anisotropy measurements. The estimated stability of the nucleotide–protein complexes has been matched with a presence of the nucleotides in the structures of the proposed nucleotide–protein complexes. It has been shown that the ribonucleotides can be definitely identified in the experimental electron density maps at equilibrium dissociation constant <10 μM. At K_D of 20–40 μM, long incubation of the protein crystals in the nucleotide solution is required to obtain the structures of the complexes. The complexes with K_D value higher than 50 μM are not stable enough to survive in crystallization conditions.     

Adenovirus virus-associated RNAII-derived small RNAs are efficiently incorporated into the rna-induced silencing complex and associate with polyribosomes

Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3' strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.     

Study on the interactions between anti-HIV-1 active compounds with trans-activation response RNA by affinity capillary electrophoresis

The study on the interactions between two anti-human immunodeficiency virus type 1 (anti-HIV-1) active compounds with trans-activation response (TAR) RNA by affinity capillary electrophoresis (ACE) with UV absorbance detection is presented. The results showed that the novel active molecules could interact with TAR RNA and inhibit the reproduce process of HIV-1. The binding constants were estimated by the change of migration time of the analytes through the change of concentrations of TAR RNA in the buffer solution. The yielded binding constants of 8.87 x 10(3)M(-1) for active compound C(3) and 8.42 x 10(3)M(-1) for MC(3) at 20.0 degrees C, 0.626 x 10(3)M(-1) and 0.644 x 10(3)M(-1) at 37.0 degrees C, respectively. The thermodynamic parameters Delta H and DeltaS were obtained and shown that both hydrophobic and electrostatic interaction played roles in the binding processes. The results showed that the presented method was an easy and simple method to evaluate the interaction of small molecules with some bioactive materials.     

Maintenance of pBR322-derived plasmids without functional RNAI

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.     

Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure

Efficient gene control by antisense RNA requires rapid bi-molecular interaction with a cognate target RNA. A comparative analysis revealed that a YUNR motif (Y=pyrimidine, R=purine) is ubiquitous in RNA recognition loops in antisense RNA-regulated gene systems. The (Y)UNR sequence motif specifies two intraloop hydrogen bonds forming U-turn structures in many anticodon-loops and all T-loops of tRNAs, the hammerhead ribozyme and in other conserved RNA loops. This structure creates a sharp bend in the RNA phosphate-backbone and presents the following three to four bases in a solvent-exposed, stacked configuration providing a scaffold for rapid interaction with complementary RNA. Sok antisense RNA from plasmid R1 inhibits translation of the hok mRNA by preventing ribosome entry at the mok Shine & Dalgarno element. The 5' single-stranded region of Sok-RNA recognizes a loop in the hok mRNA. We show here, that the initial pairing between Sok antisense RNA and its target in hok mRNA occurs with an observed second-order rate-constant of 2 x 10(6) M(-1) s(-1). Mutations that eliminate the YUNR motif in the target loop of hok mRNA resulted in reduced antisense RNA pairing kinetics, whereas mutations maintaining the YUNR motif were silent. In addition, RNA phosphate-backbone accessibility probing by ethylnitrosourea was consistent with a U-turn structure formation promoted by the YUNR motif. Since the YUNR U-turn motif is present in the recognition units of many antisense/target pairs, the motif is likely to be a generally employed enhancer of RNA pairing rates. This suggestion is consistent with the re-interpretation of the mutational analyses of several antisense control systems including RNAI/RNAII of ColE1, CopA/CopT of R1 and RNA-IN/RNA-OUT of IS10.     

Equilibrium and kinetic studies of the interactions of a porphyrin with low-density lipoproteins

Low-density lipoproteins (LDL) play a key role in the delivery of photosensitizers to tumor cells in photodynamic therapy. The interaction of deuteroporphyrin, an amphiphilic porphyrin, with LDL is examined at equilibrium and the kinetics of association/dissociation are determined by stopped-flow. Changes in apoprotein and porphyrin fluorescence suggest two classes of bound porphyrins. The first class, characterized by tryptophan fluorescence quenching, involves four well-defined sites. The affinity constant per site is 8.75 x 10(7) M(-1) (cumulative affinity 3.5 x 10(8) M(-1)). The second class corresponds to the incorporation of up to 50 molecules into the outer lipidic layer of LDL with an affinity constant of 2 x 10(8) M(-1). Stopped-flow experiments involving direct LDL porphyrin mixing or porphyrin transfer from preloaded LDL to albumin provide kinetic characterization of the two classes. The rate constants for dissociation of the first and second classes are 5.8 and 15 s(-1); the association rate constants are 5 x 10(8) M(-1) s(-1) per site and 3 x 10(9) M(-1) s(-1), respectively. Both fluorescence and kinetic analysis indicate that the first class involves regions at the boundary between lipids and the apoprotein. The kinetics of porphyrin-LDL interactions indicates that changes in the distribution of photosensitizers among various carriers could be very sensitive to the specific tumor microenvironment.     

Interaction of wheat germ translation initiation factor 2 with GDP and GTP.

The wheat germ translation initiation factor 2 (WGeIF-2) was isolated in a homogeneous state by an efficient procedure and characterized. Its molecular mass, as determined by a gel-filtration method is approximately 150,000 Da. According to SDS-PAGE WGeIF-2 consists of four subunits with M(r) 37,000 (alpha), 40,000 (beta), 42,000 (gamma) and 52,000 (delta). The beta- and gamma-subunits (but not the alpha-subunit) of WGeIF-2 can be readily phosphorylated by the double-stranded RNA activated kinase isolated from rabbit reticulocytes. Dissociation constants for WGeIF-2 complexes with GDP and GTP were measured. In our evaluation the WGeIF-2 affinity for GDP (KdGDP = 1.5 x 10(-7) M) was only 10 times higher than for GTP (KdGTP = 1.5 x 10(-6) M), while for rabbit reticulocyte eIF-2 (RReIF-2) the difference has been estimated as as much as two orders of magnitude in accordance with the literature. Close values of dissociation constants for WGeIF-2 complexes with guanine nucleotides suggest that at a sufficiently high [GTP]/[GDP] ratio the nucleotide exchange in wheat cells may take place without the participation of specific factor (eIF-2B) which catalyzes the nucleotide exchange on eIF-2 from mammalian cells.