Expression and purification of the predicted PEG10 aspartyl protease domain

Paternally Expressed Gene 10 (PEG10) is an imprinted, retrotransposon-derived gene found in mammals. Although many of the retrotransposon domains have become degenerated in PEG10, a predicted retroviral-type aspartyl protease (AP) domain has been highly conserved. Retroviral-type APs play a crucial role in the replication of some retroviruses such as the Human Immunodeficiency Virus (HIV) and are there fore important drug targets. Consequently, extensive biochemical and structural data are available for this class of proteins, although the vast majority of this has been gathered from only a small number of retroviral enzymes. Preliminary evidence indicates that the PEG10 AP is an active protease, although proteolysis by this enzyme has yet to be observed in vitro (Clark et al., 2007). This study aimed to express, purify, and characterise the predicted PEG10 AP. A number of PEG10 AP clones, each with different termini and across more than one recombinant expression system, were expressed to produce the PEG10 AP domain in E. coli. The majority of expressed proteins were largely insoluble and unsuitable for further characterisation. One clone, however, produced soluble PEG10 AP in sufficient quantities for purification and further analysis. Several lines of evidence indicated that the purified protein was dimeric in solution, consistent with the quaternary structure of other retroviral-type APs. The results presented in this thesis support the hypothesis that the PEG10 AP is active and has retained characteristics from the ancestral retrotransposon enzyme. The expression and purification protocol that has been developed can now be used to generate PEG10 AP for detailed biophysical and functional characterisation. Widely used protease inhibitors used for treatment of infection with HIV have pleiotropic effects in patients and are known to inhibit a diverse range of proteases. The development of an in vitro activity assay and testing whether the PEG10 AP is a serendipitous target of these inhibiting compounds will not only enhance our understanding of retroviral-type APs but potentially contribute to their use as therapeutic agents

RqcH and RqcP catalyze processive poly-alanine synthesis in a reconstituted ribosome-associated quality control system

In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway

Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ(2) mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery)

The Highly Conserved Codon following the Slippery Sequence Supports −1 Frameshift Efficiency at the HIV-1 Frameshift Site

<div><p>HIV-1 utilises −1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating −1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the ‘intercodon’) contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules—eRF1 protein or a cognate suppressor tRNA—were able to access and decode the intercodon prior to −1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.</p></div

The identity of the intercodon influences −1 frameshift efficiency.

<p>Top: schematic representations of the base of the HIV-1 stem-loop, with the natural glycine codon and variants (left) and the substituted stop codon and variants showing the intercodon (grey) and any complementary stem-loop alterations (bold). The end of the slippery sequence is underlined. Below: frameshift efficiency of the natural intercodon and variants as assayed with the dual luciferase reporter system. GGG_AA and UGA_U indicate the intercodon (left part) and the modification to the complementary sequences in the stem-loop (right part). The mean ± standard error of the mean (SEM) for 18 replicates from three individual experiments in COS-7 cells is shown. ***<i>P</i> = < 0.001 compared to the GGG intercodon.</p

Specific intercodon suppressor tRNAs affect recoding efficiency.

<p>A. Readthrough at the test stop signals UAGAAG, UGAAAG and UAAAAG in HEK293T cells with cognate or non-cognate suppressor tRNAs (UAG [black], UGA [grey] or UAA [white]) and tRNA^Ser as a control (serine). The mean ± SEM for four replicates is shown. B. Effect of over-expressing cognate suppressor tRNA^UGA, non-cognate tRNA^UAG or control tRNA^Ser on human antizyme +1 frameshift efficiency. The mean ± SEM for four replicates is shown. C. Effect on −1 frameshift efficiency of the HIV-1 element when UAG (black) or UGA (grey) suppressor tRNAs or the control tRNA^Ser (white) are expressed. Frameshift efficiency was measured for the cognate, non-cognate, and control tRNAs. Note the change in scales between when the intercodon is the native GGG (left) and UGA or UGA with a complementary mutation restoring the stem (UGA_U, right). The mean ± SEM for four replicates (GGG) or six replicates (UGA and UGA_U) is shown. *<i>P</i> = < 0.05, **<i>P</i> = < 0.01, ***<i>P</i> = < 0.001.</p

A modified model for −1 frameshifting in HIV-1.

<p>A. The first nucleotide (U) of the slippery sequence (blue), as part of the ‘BCX’ codon [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.ref026" target="_blank">26</a>], is positioned in the A site. The extended stem-loop is at the entrance to the ribosome, with the first nucleotide of the intercodon (orange) in the +13 position. B. The lower stem of the structure unwinds and allows the slippery sequence into the decoding centre. The stable upper stem-loop is positioned near the entry channel of the ribosome. Tension arising from resistance to unwinding may allow slippage from the A and P sites at this stage. C. After partial unwinding of the stable upper stem-loop, binding of tRNA^Gly to the intercodon, perhaps in a distorted state, competes with −1 frameshifting. D. The tRNA^Gly bound to the intercodon is decoded and translation proceeds in the 0 frame. This is the most common event, resulting in the translation of the Gag product. E. If the tRNA^Gly is not decoded, −1 frameshifting by the E and P site tRNAs occurs to relieve tension on the mRNA, resulting in the translation of Gag-Pol. An incoming tRNA^Arg is shown.</p

The effect of eRF1 depletion on termination and recoding.

<p>A. eRF1 transcript levels measured using quantitative real-time PCR. Results show eRF1 transcripts within RNA isolated from non-transfected HEK293T cells (‘none’), and eRF1 transcripts within RNA isolated from those transfected with α-eRF1 or negative control (−) vectors containing shRNAs. The mean ± standard deviation (SD) for six replicates is shown. B. Immunoblot of eRF1 in protein extracts from non-transfected HEK293T cells (‘none’), or cells transfected with α-eRF1 or negative control (−) vectors containing shRNAs. The ratios were calculated after normalisation to β-actin in each case and compared with the non-transfected control (1.0). Raw data is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.s001" target="_blank">S1 Fig.</a> C. Readthrough at a UGA test context (UGACAG). A dual luciferase construct with the two reporters in the same frame and the test stop signal separating them was co-transfected with the control and α-eRF1 shRNAs. Readthrough at the test stop signal was determined in each case (α-eRF1 and − control.) The mean ± SEM for 12 replicates from three individual experiments is shown. D. Effect of depletion of eRF1 on +1 frameshift efficiency at the human antizyme frameshift element. The mean ± SEM for eight replicates is shown. E. Effect of eRF1 depletion on frameshifting with the native GGG intercodon and substituted stop codon (UGA or UAG). The mean ± SEM for a minimum of 10 replicates from at least two independent experiments is shown. A dotted line indicates the level of −1 PRF in the absence of any shRNA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.g002" target="_blank">Fig. 2</a>). **<i>P</i> = < 0.01, ***<i>P</i> = < 0.001, n.s., not significant.</p

eRF1 over-expression influences frameshifting.

<p>A. eRF1 transcript levels measured using quantitative real-time PCR. Results show non-transfected HEK293T cells (‘none’), and those transfected with pcDNA-eRF1 vector or pcDNA3.1(+) with no insert (empty vector). The mean ± SD for six replicates is shown. B. Immunoblot of eRF1 expression in non-transfected HEK293T cells (‘none’), or cells transfected with pcDNA3.1(+) with no insert (empty vector) or pcDNA-eRF1 (eRF1). The ratios were calculated after normalisation to β-actin and comparison with the non-transfected control (1.0). Raw data are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122176#pone.0122176.s003" target="_blank">S3 Figs.</a> C. Effect of over-expression of eRF1 on readthrough at UGA test contexts. The mean ± SEM for four replicates (UGAAAG) or 12 replicates from three individual experiments (UGACUG) is shown. Cells were transfected with either empty vector (black) or pcDNA-eRF1 (grey). Values are shown above the bars for the strong stop signal UGAAAG. D. Effect of over-expression of eRF1 on +1 frameshift efficiency at the human antizyme frameshift element. The mean ± SEM for six replicates is shown. E. Effect of over-expression of eRF1 on HIV-1 −1 frameshift efficiency with GGG intercodon and when it is substituted with a stop codon (UGA or UAG). The mean ± SEM for six replicates is shown. pcDNA (black), pcDNA-eRF1 (grey). *<i>P</i> = < 0.05, **<i>P</i> = < 0.01, ***<i>P</i> = < 0.001, n.s., not significant.</p

Frameshift efficiency is dependent on the identity of the first nucleotide of the intercodon.

<p>Frameshift efficiency for the NGG (black) and NGA (grey) contexts show the mean ± SEM for 33 replicates from five individual experiments in HEK293T cells.</p