Dynamic Motions of the HIV-1 Frameshift Site RNA

The HIV-1 frameshift site (FS) plays a critical role in viral replication. During translation, the HIV-1 FS transitions from a 3-helix to a 2-helix junction RNA secondary structure. The 2-helix junction structure contains a GGA bulge, and purine-rich bulges are common motifs in RNA secondary structure. Here, we investigate the dynamics of the HIV-1 FS 2-helix junction RNA. Interhelical motions were studied under different ionic conditions using NMR order tensor analysis of residual dipolar couplings. In 150 mM potassium, the RNA adopts a 43°(±4°) interhelical bend angle (β) and displays large amplitude, anisotropic interhelical motions characterized by a 0.52(±0.04) internal generalized degree of order (GDOint) and distinct order tensor asymmetries for its two helices (η = 0.26(±0.04) and 0.5(±0.1)). These motions are effectively quenched by addition of 2 mM magnesium (GDOint = 0.87(±0.06)), which promotes a near-coaxial conformation (β = 15°(±6°)) of the two helices. Base stacking in the bulge was investigated using the fluorescent purine analog 2-aminopurine. These results indicate that magnesium stabilizes extrahelical conformations of the bulge nucleotides, thereby promoting coaxial stacking of helices. These results are highly similar to previous studies of the HIV transactivation response RNA, despite a complete lack of sequence similarity between the two RNAs. Thus, the conformational space of these RNAs is largely determined by the topology of their interhelical junctions

Thermostability and excision activity of polymorphic forms of hOGG1

Abstract Objectives Reactive oxygen species (ROS) oxidize guanine residues in DNA to form 7,8-dihydro-oxo-2′-deoxyguanosine (8oxoG) lesions in the genome. Human 8-oxoguanine glycosylase-1 (hOGG1) recognizes and excises this highly mutagenic species when it is base-paired opposite a cytosine. We sought to characterize biochemically several hOGG1 variants that have been found in cancer tissues and cell lines, reasoning that if these variants have reduced repair capabilities, they could lead to an increased chance of mutagenesis and carcinogenesis. Results We have over-expressed and purified the R46Q, A85S, R154H, and S232T hOGG1 variants and have investigated their repair efficiency and thermostability. The hOGG1 variants showed only minor perturbations in the kinetics of 8oxoG excision relative to wild-type hOGG1. Thermal denaturation monitored by circular dichroism revealed that R46Q hOGG1 had a significantly lower Tm (36.6 °C) compared to the other hOGG1 variants (40.9 °C to 43.2 °C). Prolonged pre-incubation at 37 °C prior to the glycosylase assay dramatically reduces the excision activity of R46Q hOGG1, has a modest effect on wild-type hOGG1, and a negligible effect on A85S, R154H, and S232T hOGG1. The observed thermolability of hOGG1 variants was mostly alleviated by co-incubation with stoichiometric amounts of competitor DNA

Ribosomal Frameshifting in HTLV-1: Examining the pro-pol frameshift site

Human t-cell lymphotropic virus type l (HTLV-1) was the first identified human retrovirus, identified in 1980. Infection with HTLV-1 results in adult T-cell leukemia with 5-10% incidence. An estimated 15-20 million individuals worldwide are infected with HTLV. Replication of retroviruses, such as HTLV, is dependent upon synthesis of viral structural and enzymatic proteins. Synthesis of HTLV’s enzymatic proteins (Protease (PR), Reverse Transcriptase (RT), and Integrase (IN)) is dependent upon programmed ribosomal frameshifting (PRF). PRF is defined as a programmed change in the ribosome’s reading frame during translation. HTLV-1 has been observed to have ribosomal frameshifting at two difference sites. The frameshift sites gag-pro and pro-pol have been established but the efficiencies and structures of these two frameshift sites has not yet been determined. The HTLV-1 pro-pol site consists of three RNA elements: a slippery sequence (UUUAAAC), a spacer, and a downstream structure. In this work, the HTLV pro-pol -1PRF mechanism is investigated. A pseudoknot structure is predicted downstream of the slippery sequence. We hypothesize the pseudoknot structure contributes significantly to the frameshift efficiency. To test this hypothesis, we designed four variant frameshift sites to test the importance of the pseudoknot structure to frameshifting. An in vitro dual-luciferase frameshift assay was utilized to determine the frameshift efficiencies for the wild-type and variant frameshift sites

Structure and Dynamics of the HIV‑1 Frameshift Element RNA

The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target. The prior model for this motif contains two helices separated by a three-nucleotide bulge. Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features of the SHAPE-directed model include alternate helical conformations and a larger, more complex structure. These structural elements also support the presence of a secondary frameshift site within the frameshift domain. Here, we use oligonucleotide-directed structure perturbation, probing in the presence of formamide, and in-virion experiments to examine these models. Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain. Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation. Translational frameshifting assays with frameshift domain mutants support a functional role for the interactions predicted by and specific to the SHAPE-directed model. These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs