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Durney, Michael

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Durney

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Durney, Michael
Author's Publications: search.filters.isAuthorOfPublication.660e65e8-9eb2-42de-8875-8f910acfb6b9

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    Preformed Protein-Binding Motifs in 7SK snRNA: Structural and Thermodynamic Comparisons with Retroviral TAR
    (Elsevier, 2010) Durney, Michael; D'Souza, Victoria
    The 7SK small nuclear RNA is a highly conserved non-coding RNA that regulates transcriptional elongation. 7SK utilizes the HEXIM proteins to sequester the transcription factor P-TEFb by a mechanism similar to that used by retroviral TAR RNA to engage Tat and P-TEFb. Tat has also recently been shown to bind 7SK directly and recruit P-TEFb to TAR. We report here the solution structures of the free and arginine-bound forms of stem loop 4 of 7SK (7SK-SL4). Comparison of the 7SK-SL4 and TAR structures demonstrates the presence of a common arginine sandwich motif. However, arginine binding to 7SK-SL4 is mechanistically distinct and occurs via docking into a pre-organized pocket resulting in a 1000-fold increased affinity. Furthermore, whereas formation of the binding pocket in TAR requires a critical base-triple, hydrogen-bond formation between the equivalent bases in 7SK-SL4 is not essential and the pocket is stabilized solely by a pseudo base-triple platform. In addition, this theme of preformed protein binding motifs also extends into the pentaloop. The configuration of the loop suggests that 7SK-SL4 is poised to make ternary contacts with P-TEFb and HEXIM or Tat. These key differences between 7SK-SL4 and TAR present an opportunity to understand RNA structural adaptation and have implications for understanding differential interactions with Tat.
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    An Equilibrium-Dependent Retroviral mRNA Switch Regulates Translational Recoding
    (Nature Publishing Group, 2011) Houck-Loomis, Brian; Durney, Michael; Salguero, Carolina; Shankar, Neelaabh; Nagle, Julia Marie; Goff, Stephen; D'Souza, Victoria
    Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag–Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5–10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag–Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.