Biomedical subjects

J Stephen Lodmell

Publications and source records attributed to J Stephen Lodmell.

12 recordsLinked to original sources

PubMed2007

An extended stem-loop 1 is necessary for human immunodeficiency virus type 2 replication and affects genomic RNA encapsidation.

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer. In this study, we demonstrate that a 10-nucleotide palindromic sequence (pal) located at the 3' end of the psi encapsidation signal is critical for human immunodeficiency virus type 2 (HIV-2) replication and affects genomic RNA encapsidation. We used short-term and long-term culture of pal-mutated viruses in permissive C8166 cells and their phenotypic reversion to show the existence of a structurally extended SL1 during HIV-2 replication, formed by the interaction of the 3' end of the pal within psi with a motif located downstream of SL1. The stem extending HIV-2 SL1 is structurally similar to stem B described for HIV-1 SL1. Despite the high degree of phylogenetic conservation, these results show that mutant viruses are viable when the autocomplementary nature of the pal sequence is disrupted, but not without a stable stem B. Our observations show that formation of the extended SL1 is necessary during viral replication and positively affects HIV-2 genomic RNA encapsidation. Sequestration of part of the packaging signal into SL1 may be a means of regulating its presentation during the replication cycle.

Animals↗

PubMed2006

Mutations conferring aminoglycoside and spectinomycin resistance in Borrelia burgdorferi.

We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 x 10(-6). These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 x 10(-9) to 1 x10(-7) in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

Aminoglycosides↗

PubMed2005

A time-resolved investigation of ribosomal subunit association.

The notion that the ribosome is dynamic has been supported by various biochemical techniques, as well as by differences observed in high-resolution structures of ribosomal complexes frozen in various functional states. Yet, the mechanisms and extent of rRNA dynamics are still largely unknown. We have used a novel, fast chemical-modification technique to provide time-resolved details of 16 S rRNA structural changes that occur as bridges are formed between the ribosomal subunits as they associate. Association of different 16 S rRNA regions was found to be a sequential, multi-step process involving conformational rearrangements within the 30 S subunit. Our results suggest that key regions of 16 S rRNA, necessary for decoding and tRNA A-site binding, are structurally altered in a time-dependent manner by association with the 50 S ribosomal subunits.

Base Pairing↗

PubMed2005

Ribosome builder: a software project to simulate the ribosome.

The Ribosome Builder is a software project that provides tools and techniques to create dynamic models of macromolecular systems from the rapidly growing numbers of atomic structural models. It includes a computer program that allows the user to assemble the multiple molecular components within a 3D space and to define the hypothetical interactions of these components with the initial goal of understanding protein translation at an atomic level of detail. The program employs a simplified molecular dynamics forcefield that can simulate the long time-scale events, such as docking of translation factors and mRNA translocation. An embedded scripting language and Application Programming Interface (API) enable the creation of Steered Molecular Dynamics (SMD) simulations through the programmable application of external forces and torques on atoms and bonds. A graphical interface is provided for displaying and interacting with models, recording movies of molecular dynamics movements, and creating annotated 3D simulations of complex macromolecular events. Initial applications of the project include simulation of tetraloop folding, docking of an mRNA on the 30S subunit and a schematic simulation of the translation elongation cycle. The program is an open source project released under the GNU public license.

Computer Simulation↗

PubMed2004

Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain.

Ribosomal protein L11 and the L11 binding region of ribosomal RNA constitute an important domain involved in active functions of the ribosome during translation. We studied the effects of L11 knock-out and truncation mutations on the structure of the rRNA in this region and on its interactions with a translation elongation factor and the antibiotic thiostrepton. The results indicated that the structure of the L11-binding rRNA becomes conformationally flexible when ribosomes lack the entire L11 protein, but not when the C-terminal domain is present on ribosomes. Probing wild type and mutant ribosomes in the presence of the antibiotic thiostrepton and elongation factor-G (EF-G) rigorously localized the binding cleft of thiostrepton and suggested a role for the rRNA in the L11-binding domain in modulating factor binding. Our results also provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize EF-G binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction. The implications for recent models of thiostrepton activity and factor interactions are discussed.

Anti-Bacterial Agents↗

PubMed2004

Splicing affects presentation of RNA dimerization signals in HIV-2 in vitro.

During retroviral replication, full-length viral RNAs are encapsidated into new virus particles, while spliced RNAs are excluded. The Retroviridae are unique among viruses in that infectious viral particles contain a dimer of two identical genomic RNA strands. A variety of experimental data has suggested that dimerization and encapsidation of full-length viral RNAs are linked processes, although whether dimerization is a prerequisite for encapsidation, or conversely, dimerization follows encapsidation, has not been firmly established. If dimerization was the sole determinant for encapsidation, then spliced viral RNAs might be expected to display a reduced capacity for dimerization, resulting in their exclusion from the dimerization pool. Here, we studied the in vitro dimerization properties of unspliced and spliced HIV-2 RNA. We find that the rate and yield of dimerization of Nef, Rev and Tat spliced RNAs exceeded those of unspliced RNA. Although these data do not support a simple correlation between dimerization efficiency and the presence of introns, they establish that splicing affects the presentation of dimerization signal(s), which we corroborate with structure probing. This change in RNA conformation likely affects the RNA's suitability for packaging. Furthermore, the presence of upstream and downstream elements that affect the conformation of the packaging signal represents a potentially efficient viral strategy for correctly sorting spliced versus unspliced RNAs.

Base Sequence↗

PubMed2004

The translation initiation functions of IF2: targets for thiostrepton inhibition.

Bacterial translation initiation factor IF2 was localized on the ribosome by rRNA cleavage using free Cu(II):1,10-orthophenanthroline. The results indicated proximity of IF2 to helix 89, to the sarcin-ricin loop and to helices 43 and 44, which constitute the "L11/thiostrepton" stem-loops of 23S rRNA. These findings prompted an investigation of the L11 contribution to IF2 activity and a re-examination of the controversial issue of the effect on IF2 functions of thiostrepton, a peptide antibiotic known primarily as a powerful inhibitor of translocation. Ribosomes lacking L11 were found to have wild-type capacity to bind IF2 but a strongly reduced ability to elicit its GTPase activity. We found that thiostrepton caused a faster recycling of this factor on and off the 70S ribosomes and 50S subunits, which in turn resulted in an increased rate of the multiple turnover IF2-dependent GTPase. Although thiostrepton did not inhibit the P-site binding of fMet-tRNA, the A-site binding of the EF-Tu-GTP-Phe-tRNA or the activity of the ribosomal peptidyl transferase center (as measured by the formation of fMet-puromycin), it severely inhibited IF2-dependent initiation dipeptide formation. This inhibition can probably be traced back to a thiostrepton-induced distortion of the ribosomal-binding site of IF2, which leads to a non-productive interaction between the ribosome and the aminoacyl-tRNA substrates of the peptidyl transferase reaction. Overall, our data indicate that the translation initiation function of IF2 is as sensitive as the translocation function of EF-G to thiostrepton inhibition.

Binding Sites↗

PubMed2003

Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro.

An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1, a 5' leader region site termed stem-loop 1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5'-end of the primer-binding site (PBS) and a stem-loop region homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization and electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5'- and/or 3'-ends and by making compensatory base substitutions, we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1-dependent tight dimers, probably by sequestering SL1 in a stable intramolecular arrangement. Moreover, we found that nucleotides downstream of SL1 decreased the rate of tight dimerization. Interestingly, dimerization at 37 degrees C in the presence of nucleocapsid protein increased the yield of SL1-mediated tight dimerization in vitro, even in the presence of the two interfering elements, suggesting a relationship between the nucleocapsid protein and activation of the SL1 dimerization signal in vivo.

3' Untranslated Regions↗

PubMed2003

Elucidation and characterization of oligonucleotide-accessible sites on HIV-2 leader region RNA.

The retroviruses, including the human pathogens HIV-1 and HIV-2, are diploid inasmuch as they encapsidate two copies of their RNA genome. Prior to or during encapsidation, two copies of full-length genomic RNA recognize and stably bind each other in a process called dimerization. RNA structures within the viral genome promote dimerization in both HIV-1 and HIV-2 and are located in the 5'-untranslated leader region. Inhibition of dimerization by mutation of these RNA signals has been demonstrated to drastically reduce viral infectivity and replication kinetics and, thus, represents a potential target for antiretroviral therapy. In this study, we identified sites in HIV-2 leader region RNA that are functionally accessible to hybridization with oligonucleotides (ODNs) by reverse transcription with random ODN libraries (RT-ROL). We then tested specific ODNs directed against these regions for their efficacy in inhibiting RNA dimerization in vitro. We determined that of several hybridization-competent ODNs, only two were very effective in inhibiting RNA dimerization. Both of these ODNs were complementary to viral RNA at the primer binding site (PBS). These results identify regions with high accessibility to ODN binding on HIV-2 RNA and help to map the region(s) essential for dimerization within the viral RNA.

5' Untranslated Regions↗

PubMed2003

Ribosomal localization of translation initiation factor IF2.

Bacterial translation initiation factor IF2 is a GTP-binding protein that catalyzes binding of initiator fMet-tRNA in the ribosomal P site. The topographical localization of IF2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. Here, we present a model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II):EDTA tethered to cysteine residues introduced into IF2. Two specific amino acids in the GII domain of IF2 are in proximity to helices H3, H4, H17, and H18 of 16S rRNA. Furthermore, the junction of the C-1 and C-2 domains is in proximity to H89 and the thiostrepton region of 23S rRNA. The docking is further constrained by the requisite proximity of the C-2 domain with P-site-bound tRNA and by the conserved GI domain of the IF2 with the large subunit's factor-binding center. Comparison of our present findings with previous data further suggests that the IF2 orientation on the 30S subunit changes during the transition from the 30S to 70S initiation complex.

Base Sequence↗

PubMed2003

A structural linkage between the dimerization and encapsidation signals in HIV-2 leader RNA.

The 5' untranslated leader region of retroviral RNAs contains noncoding information that is essential for viral replication, including signals for transcriptional transactivation, splicing, primer binding for reverse transcription, dimerization of the genomic RNA, and encapsidation of the viral RNA into virions. These RNA motifs have considerable structural and functional overlap. In this study, we investigate the conformational dynamics associated with the use and silencing of a sequence in HIV-2 RNA that is involved in genomic RNA dimerization called stem-loop 1 (SL1) and its relationship with a flanking sequence that is known to be important for encapsidation of viral RNAs. We demonstrate that a long-distance intramolecular interaction between nucleotides located upstream of the primer-binding site domain and nucleotides encompassing the Gag translation start codon functionally silences SL1 as a dimerization element. This silencing can be relieved by mutation or by hybridization of an oligonucleotide that disrupts the long-distance interaction. Furthermore, we identify a palindrome within the packaging/encapsidation signal Psi (just 5' of SL1) that can either serve as an efficient dimerization signal itself, or can mediate SL1 silencing through base pairing with SL1. These results provide a tangible link between the functions of genomic RNA dimerization and encapsidation, which are known to be related, but whose physical relationship has been unclear. A model is proposed that accounts for observations of dimerization, packaging, and translation of viral RNAs during different phases of the viral replication cycle.

Base Sequence↗

PubMed2002

Alternate usage of two dimerization initiation sites in HIV-2 viral RNA in vitro.

An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during encapsidation and budding. In HIV-1, a stem-loop structure in the genomic RNA called the dimerization initiation site, or DIS, has been well characterized. However, the identification of the structure(s) necessary for dimerization of HIV-2 genomic RNA has been less straightforward, as reflected by recent conflicting reports. Here, using a variety of mutant and wild-type RNA constructs and a systematic analysis of experimental conditions, we demonstrate that two dimerization sites in HIV-2 RNA are clearly discernible under different experimental conditions. A short sequence overlapping the primer binding site acts as the default dimerization site for wild-type viral RNA transcripts of several lengths provided that dimerization incubation conditions do not include a high heat step (>50 degrees C), and electrophoresis is carried out under mild conditions that do not deplete the RNA of magnesium. However, some RNA constructs are able to dimerize through stem-loop 1 (SL1), which is the structure homologous to the HIV-1 DIS, under certain experimental conditions. Interestingly, deletion or mutation of the default PBS dimerization site leads to efficient usage of the SL1 dimerization site. This study defines conditions under which each site may be used for dimerization and demonstrates, furthermore, the facility with which the two sites can substitute for each other. This is suggestive of a switching mechanism that may be used in the viral replication cycle.

Base Sequence↗