Biomedical subjects
Publications and source records attributed to Rachel Green.
The Protein Data Bank (PDB; http://www.pdb.org) is the primary source of information on the 3D structure of biological macromolecules. The PDB's mandate is to disseminate this information in the most usable form and as widely as possible. The current query and distribution system is described and an alpha version of the future re-engineered system introduced.
Evolutionary approaches are regularly used to isolate single molecules with desired activities from large populations of nucleic acids (approximately 10(15)). Several methods have also been developed to generate libraries of mRNA-encoded peptides and proteins for the in vitro selection of functional polypeptides. In principal, such mRNA encoding systems could be used with libraries of nonbiological polymers if the ribosome can be directed to polymerize tRNAs carrying unnatural amino acids. The fundamental problem is that current chemical aminoacylation systems cannot easily produce sufficient amounts of the numerous misacylated tRNAs required to synthesize a complex library of encoded polymers. Here, we show that bulk-aminoacylated tRNA can be transformed into N-monomethylated aminoacyl tRNA and translated. Because poly-N-methyl peptide backbones are refractory to proteases and are membrane permeable, our method provides an uncomplicated means of evolving novel drug candidates.
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Following peptide-bond formation, the mRNA:tRNA complex must be translocated within the ribosomal cavity before the next aminoacyl tRNA can be accommodated in the A site. Previous studies suggested that following peptide-bond formation and prior to EF-G recognition, the tRNAs occupy an intermediate (hybrid) state of binding where the acceptor ends of the tRNAs are shifted to their next sites of occupancy (the E and P sites) on the large ribosomal subunit, but where their anticodon ends (and associated mRNA) remain fixed in their prepeptidyl transferase binding states (the P and A sites) on the small subunit. Here we show that pre-translocation-state ribosomes carrying a dipeptidyl-tRNA substrate efficiently react with the minimal A-site substrate puromycin and that following this reaction, the pre-translocation-state bound deacylated tRNA:mRNA complex remains untranslocated. These data establish that pre-translocation-state ribosomes must sample or reside in an intermediate state of tRNA binding independent of the action of EF-G.
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The collection of peripheral blood progenitor cells (PBPC) by apheresis has become common in related allogeneic donors. However, the acceptability of the procedure to donors has not been documented. The purpose of this baseline case series study was to evaluate the psycho-social dimensions of apheresis from the perspective of healthy sibling donors and to explore issues surrounding fully informed consent including voluntary donation. At the first interview to discuss donation, 17 consecutive human leucocyte antigens (HLA) identical sibling donors who chose to donate PBPC were recruited to the study. They then completed both scales of the State-Trait Anxiety Inventory. The state scale was completed again immediately before first apheresis. At the end of the final apheresis, the donors were interviewed again by an independent researcher using a standardised questionnaire. All aspects of the procedure were well tolerated, including levels of anxiety and pain. Donors donated even if the relationship with their sibling was poor. However, some areas for improvement were highlighted. Eight (47%) donors were asked to donate by their sibling or another close relative, and this gave them no real volunteer status. Written information was judged important by 11 (65%) donors, but the material used was limited. The possibility of a poor outcome for the recipient was not well understood. The content of the written documentation and the management of confidentiality in terms of donor volunteer status needed to be addressed. A further study regarding the follow-up needs of donors, including those where the outcome is poor, is underway.
Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (EF-G) during the translation cycle. Previous studies established that modification of ribosomal proteins with thiol-specific reagents promotes this event in the absence of EF-G. Here we identify two small subunit interface proteins S12 and S13 that are essential for maintenance of a pretranslocation state. Omission of these proteins using in vitro reconstitution procedures yields ribosomal particles that translate in the absence of enzymatic factors. Conversely, replacement of cysteine residues in these two proteins yields ribosomal particles that are refractive to stimulation with thiol-modifying reagents. These data support a model where S12 and S13 function as control elements for the more ancient rRNA- and tRNA-driven movements of translocation.
Conventional highly active antiretroviral therapy (HAART) regimens used to treat human immunodeficiency virus (HIV) infection typically use nucleoside reverse transcriptase inhibitors (NRTIs) and either a protease inhibitor (PI) or a non-nucleoside reverse transcriptase inhibitor (NNRTI). Because PI-based regimens are associated with significant long-term toxicity and adherence difficulty, there is a need for novel regimens that maximize combination treatment options. This 12-month, observational, cohort study evaluated the efficacy, safety, and tolerability of a novel three-drug HAART regimen. Drug treatment consisted of nevirapine (NVP), efavirenz (EFV), and didanosine (ddl). Twenty-six treatment-naive and -experienced HIV-1+ men and women were included in the study. Assessment consisted of CD4+ cell count, plasma HIV-1 RNA load, and adverse effects of study medications. After one year of therapy, 11/12 treatment-naive subjects (92%) and 8/9 treatment-experienced subjects (89%) had viral loads < 400 copies/mL. Both groups also had an excellent immune response. At one year, there was a mean increase of 438 CD4+ cells/mm3 among treatment-naive subjects and 367 cells/mm3 among treatment-experienced subjects. Treatment-limiting adverse effects occurred in 3/15 treatment-naive (20%) and 2/11 treatment-experienced (18%) subjects. These preliminary data suggest that the combination of NVP, EFV, and ddl is simple, safe, and effective.
Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of the elongation factors EFG and EFTu (and the energy derived from GTP hydrolysis) is promoted by modification of the ribosome with thiol-specific reagents such as para-chloromercuribenzoate (pCMB). Here, we examine the translational cycle of modified ribosomes and show that peptide bond formation and tRNA binding are largely unaffected, whereas translocation of the mRNA:tRNA complex is substantially promoted by pCMB modification. The translocation movements that we observe are authentic by multiple criteria including the processivity of translation, accuracy of movement (three-nucleotide) along a defined mRNA template and sensitivity to antibiotics. Characterization of the modified ribosomes reveals that the protein content of the ribosomes is not depleted but that their subunit association properties are severely compromised. These data suggest that molecular targets (ribosomal proteins) in the interface region of the ribosome are critical barriers that influence the translocation of the mRNA:tRNA complex.
Recent studies are beginning to shed light on the mechanism of ribosome-catalyzed peptide bond formation.
Functional Escherichia coli 50S ribosomal subunits can be reconstituted from their natural rRNA and protein components. However, when the assembly is performed with in vitro-transcribed 23S rRNA, the reconstitution efficiency is diminished by four orders of magnitude. We tested a variety of chemical chaperones (compounds that are typically used for protein folding), putative RNA chaperones (proteins) and ribosome-targeted antibiotics (small-molecule ligands) that might be reasoned to aid in folding and assembly. Addition of the osmolyte trimethylamine-oxide (TMAO) and the ketolide antibiotic telithromycin (HMR3647) to the reconstitution stimulates its efficiency up to 100-fold yielding a substantially improved system for the in vitro analysis of mutant ribosomes.