Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation

An ultra-wide bandwidth (704 to 4 032 MHz) receiver for the Parkes radio telescope

We describe an ultra-wide-bandwidth, low-frequency receiver recently installed on the Parkes radio telescope. The receiver system provides continuous frequency coverage from 704 to 4032 MHz. For much of the band ( ${∼}60$ ), the system temperature is approximately 22 K and the receiver system remains in a linear regime even in the presence of strong mobile phone transmissions. We discuss the scientific and technical aspects of the new receiver, including its astronomical objectives, as well as the feed, receiver, digitiser, and signal processor design. We describe the pipeline routines that form the archive-ready data products and how those data files can be accessed from the archives. The system performance is quantified, including the system noise and linearity, beam shape, antenna efficiency, polarisation calibration, and timing stability

Summary of dynamic changes in closed-loop factors in translating ribosomes dependent on polysomal, polyadenylation, initiation, and elongation states.

<p>Only the translating ribosomes at initiation containing closed-loop structures are summarized. For simplicity, the dynamic changes of closed-loop factors in translating ribosomes containing other combinations of closed-loop factors are not represented, and these changes in stoichiometry are explained more completely in the text. Also, based on our results only one PAB1 is represented as binding to each poly(A) tail.</p

Relative levels of proteins in the 77S monosomal translating complex during different stages of translation.

<p>Relative levels of proteins in the 77S monosomal translating complex during different stages of translation.</p

AU-FDS analysis of two initiation conditions and polysomes.

<p>A and B. Comparison of two different initiation conditions. C-E. Expanded c(s) values to identify ribosomal-GFP protein migrations in polysomal material (greater than 90S).</p

AU-FDS and AU-A₂₃₀ analyses of extracts containing GFP fusions to translation components.

<p>For each set of data shown in Fig 1, the AUC analysis was from the same centrifuge run. Cells were grown on glucose-containing medium except as indicated. glu + - 10 min: growth was on glucose-containing medium followed by growth on medium depleted for glucose for 10 min; and glu +—+: growth was the same as glu + - 10 min except glucose was added back for 1 min. In panel A, it should be noted that the AU-A₂₃₀ abundance for the RPS4B-GFP sample was twice that of the STM1-GFP sample. A. RPL25A-Flag pull downs were conducted on strains carrying either RPS4B-GFP or STM1-GFP; B-D. Flag-PAB1 pull downs were conducted on strains carrying the GFP fusions as indicated. Data displayed in panels B and D were done on the same day on extracts split between the two centrifuge runs.</p

Analysis of Flag-SBP1 purified translation complexes.

<p>A. AU-A₂₆₀ analysis was conducted instead of AU-A₂₃₀ analysis for better detection of complexes with this particular Flag-tagged protein. A-F. Growth conditions were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150616#pone.0150616.g001" target="_blank">Fig 1</a>. C. No flag refers to a strain lacking Flag-SBP1 plasmid. The differences between the no flag control and the Flag-SBP1 pull downs in panel C-F were not considered significant for eIF4E-GFP and eIF4G2-GFP.</p

Comparison of abundance of translation factors between initiation and elongation conditions.

<p>Growth conditions for initiation conditions (glu +—+ 1 min) were obtained by adding glucose back to glucose depleted cells for 1 min, at which time cycloheximide was added and cells were harvested. Elongation conditions (glu +) were cells grown on glucose growth conditions. A-F. GFP fusions were as indicated.</p

Relative levels of proteins in the polysomal translating complexes during different stages of translation.

<p>Relative levels of proteins in the polysomal translating complexes during different stages of translation.</p

Analysis of eIF4E-Flag purified translation complexes.

<p>Growth conditions and analysis were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150616#pone.0150616.g001" target="_blank">Fig 1</a>. A and B. Strains were either transformed with eIF4E-Flag plasmid (eIF4E-Flag) or with no eIF4E plasmid (eIF4E).</p