Mass spectrometric analysis of the human 40S ribosomal subunit: Native and HCV IRES‐bound complexes

Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex

Strong Influence of Temperature and Vacuum on the Photoluminescence of In0.3Ga0.7As Buried and Surface Quantum Dots

Abstract The strong influences of temperature and vacuum on the optical properties of In0.3Ga0.7As surface quantum dots (SQDs) are systematically investigated by photoluminescence (PL) measurements. For comparison, optical properties of buried quantum dots (BQDs) are also measured. The line-width, peak wavelength, and lifetime of SQDs are significantly different from the BQDs with the temperature and vacuum varied. The differences in PL response when temperature varies are attributed to carrier transfer from the SQDs to the surface trap states. The obvious distinctions in PL response when vacuum varies are attributed to the SQDs intrinsic surface trap states inhibited by the water molecules. This research provides necessary information for device application of SQDs as surface-sensitivity sensors

Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA

Protein synthesis in all cells begins with recruitment of the small ribosomal subunit to the initiation codon in a messenger RNA. In some eukaryotic viruses, RNA upstream of the coding region forms an internal ribosome entry site (IRES) that directly binds to the 40S ribosomal subunit and enables translation initiation in the absence of many canonical translation initiation factors. The hepatitis C virus (HCV) IRES RNA requires just two initiation factors, eukaryotic initiation factor (eIF) 2 and eIF3, to form preinitiation 48S ribosomal complexes that subsequently assemble into translation-competent ribosomes. Using an RNA-based affinity purification approach, we show here that HCV IRES RNA facilitates eIF2 function through its interactions with eIF3 and the 40S ribosomal subunit. Although the wild-type IRES assembles normally into 48S and 80S ribosomal complexes in human cell extract, mutant IRES RNAs become trapped at the 48S assembly stage. Trapped 48S complexes formed by IRES mutants with reduced eIF3 binding affinity nonetheless contain eIF3, consistent with inherent eIF3-40S subunit affinity. Intriguingly, however, one of these IRES mutants prevents stable association of both eIF3 and eIF2, preventing initiator tRNA deposition and explaining the block in 80S assembly. In contrast, an IRES mutant unable to induce a conformational change in the 40S subunit, as observed previously by single-particle cryoelectron microscopy, blocks 80S formation at a later stage in assembly. These data suggest that the IRES RNA coordinates interactions of eIF3 and eIF2 on the ribosome required to position the initiator tRNA on the mRNA in the ribosomal peptidyl-tRNA site (P site)

Kinetics of Viscosity Decrease by Cellulase Treatment of Bleached Hardwood Kraft-Based Dissolving Pulp

A dissolving pulp of low cellulose viscosity represents a pulp of high quality; hence, it is often necessary to decrease the initial dissolving pulp viscosity. One so-called environmentally friendly approach to further reducing the dissolving pulp viscosity is to treat the dissolving pulp with cellulase enzymes. In this study, the kinetics of the decrease in cellulose viscosity during a cellulase treatment was investigated. The study showed that the kinetics of the cellulose degradation during a cellulase treatment can be divided into at least two phases, where the initial phase is very fast and the final phase is very slow. The kinetic two-phase model for the viscosity degradation that has been developed in this project can be used to predict and control the final pulp viscosity of dissolving pulps