Abstract:The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44–61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the i… Show more
“…The annealing activity of the full-length protein and its fragment Tat(44-61) has been tested in different assays and with different substrates (19,33). Despite earlier reports (34,35), Doetsch et al did not observe a duplex destabilization activity of the Tat protein and its fragment Tat(44-61) (19,33,36,37). We conclude that the Tat peptide accelerates annealing in a sequence-independent fashion and is thus a useful tool for the investigation of this activity.…”
Section: Resultsmentioning
confidence: 99%
“…According to Crothers (1971), the double-strand stabilization is a result of stronger peptide binding to the duplex when compared with the single-stranded form. The acceleration of annealing on the other hand is due to the selection of an annealing-competent conformation of the RNA single-strand through Tat(44-61) as we recently reported (19). …”
Section: Discussionmentioning
confidence: 95%
“…Another class of proteins, which only accelerate annealing, are called RNA annealer proteins. Also in this group electrostatic and transient interactions with the RNA are dominant and essential, as shown by the example of the HIV-1 Tat fragment Tat(44-61) (19). HIV-1 Tat is an early viral regulator whose best described function is the trans-activation of transcription through specific RNA binding, but which has been implied in many other viral processes some of which involve RNA molecules (20).…”
Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other.
“…The annealing activity of the full-length protein and its fragment Tat(44-61) has been tested in different assays and with different substrates (19,33). Despite earlier reports (34,35), Doetsch et al did not observe a duplex destabilization activity of the Tat protein and its fragment Tat(44-61) (19,33,36,37). We conclude that the Tat peptide accelerates annealing in a sequence-independent fashion and is thus a useful tool for the investigation of this activity.…”
Section: Resultsmentioning
confidence: 99%
“…According to Crothers (1971), the double-strand stabilization is a result of stronger peptide binding to the duplex when compared with the single-stranded form. The acceleration of annealing on the other hand is due to the selection of an annealing-competent conformation of the RNA single-strand through Tat(44-61) as we recently reported (19). …”
Section: Discussionmentioning
confidence: 95%
“…Another class of proteins, which only accelerate annealing, are called RNA annealer proteins. Also in this group electrostatic and transient interactions with the RNA are dominant and essential, as shown by the example of the HIV-1 Tat fragment Tat(44-61) (19). HIV-1 Tat is an early viral regulator whose best described function is the trans-activation of transcription through specific RNA binding, but which has been implied in many other viral processes some of which involve RNA molecules (20).…”
Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other.
“…(47) As a consequence, the strong acceleration of the annealing reaction between cTAR and dTAR but not TAR by Tat(44–61) confirms that this peptide promotes only annealing reactions that can already occur in the absence of peptide. Therefore, Tat(44–61) is confirmed as a nucleic acid annealer rather than a true chaperone (33). Figure 5.Evidence that Tat(44–61) does not promote cTAR/TAR RNA annealing in vitro .…”
The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44–61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44–61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer. To further understand the structure–activity relationships of this highly conserved domain, we investigated by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44–61) mutants. Our data showed that the Tyr47 and basic residues of the Tat(44–61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions, respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion.
“…where initial base pairing cannot take place. Similarly, Doetsch et al (55) suggested that a human immunodeficiency virus-1 derived Tat peptide accelerates annealing of two RNA ligands by changing the population distribution of RNA structures to favour an annealing-competent RNA conformation. Figure 6.Model of Hfq RNA chaperone function.…”
In enteric bacteria, many small regulatory RNAs (sRNAs) associate with the RNA chaperone host factor Q (Hfq) and often require the protein for regulation of target mRNAs. Previous studies suggested that the hexameric Escherichia coli Hfq (HfqEc) binds sRNAs on the proximal site, whereas the distal site has been implicated in Hfq–mRNA interactions. Employing a combination of small angle X-ray scattering, nuclear magnetic resonance and biochemical approaches, we report the structural analysis of a 1:1 complex of HfqEc with a 34-nt-long subsequence of a natural substrate sRNA, DsrA (DsrA34). This sRNA is involved in post-transcriptional regulation of the E. coli rpoS mRNA encoding the stationary phase sigma factor RpoS. The molecular envelopes of HfqEc in complex with DsrA34 revealed an overall asymmetric shape of the complex in solution with the protein maintaining its doughnut-like structure, whereas the extended DsrA34 is flexible and displays an ensemble of different spatial arrangements. These results are discussed in terms of a model, wherein the structural flexibility of RNA ligands bound to Hfq stochastically facilitates base pairing and provides the foundation for the RNA chaperone function inherent to Hfq.
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