Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells.

The processing, membrane targeting and folding of newly synthesized polypeptides is closely linked to their synthesis at the ribosome. A network of enzymes, chaperones and targeting factors engages ribosome-nascent chain complexes (RNCs) to support these maturation processes. Exploring the modes of action of this machinery is critical for our understanding of functional protein biogenesis. Selective ribosome profiling (SeRP) is a powerful method for interrogating co-translational interactions of maturation factors with RNCs. It provides proteome-wide information on the factor's nascent chain interactome, the timing of factor binding and release during the progress of translation of individual nascent chain species, and the mechanisms and features controlling factor engagement. SeRP is based on the combination of two ribosome profiling (RP) experiments performed on the same cell population. In one experiment the ribosome-protected mRNA footprints of all translating ribosomes of the cell are sequenced (total translatome), while the other experiment detects only the ribosome footprints of the subpopulation of ribosomes engaged by the factor of interest (selected translatome). The codon-specific ratio of ribosome footprint densities from selected over total translatome reports on the factor enrichment at specific nascent chains. In this chapter, we provide a detailed SeRP protocol for mammalian cells. The protocol includes instructions on cell growth and cell harvest, stabilization of factor-RNC interactions, nuclease digest and purification of (factor-engaged) monosomes, as well as preparation of cDNA libraries from ribosome footprint fragments and deep sequencing data analysis. Purification protocols of factor-engaged monosomes and experimental results are exemplified for the human ribosomal tunnel exit-binding factor Ebp1 and chaperone Hsp90, but the protocols are readily adaptable to other co-translationally acting mammalian factors.

Selective ribosome profiling reveals a role for SecB in the co-translational inner membrane protein biogenesis.

The chaperone SecB has been implicated in de novo protein folding and translocation across the membrane, but it remains unclear which nascent polypeptides SecB binds, when during translation SecB acts, how SecB function is coordinated with other chaperones and targeting factors, and how polypeptide engagement contributes to protein biogenesis. Using selective ribosome profiling, we show that SecB binds many nascent cytoplasmic and translocated proteins generally late during translation and controlled by the chaperone trigger factor. Revealing an uncharted role in co-translational translocation, inner membrane proteins (IMPs) are the most prominent nascent SecB interactors. Unlike other substrates, IMPs are bound early during translation, following the membrane targeting by the signal recognition particle. SecB remains bound until translation is terminated, and contributes to membrane insertion. Our study establishes a role of SecB in the co-translational maturation of proteins from all cellular compartments and functionally implicates cytosolic chaperones in membrane protein biogenesis.

Mechanisms of Cotranslational Protein Maturation in Bacteria.

Growing cells invest a significant part of their biosynthetic capacity into the production of proteins. To become functional, newly-synthesized proteins must be N-terminally processed, folded and often translocated to other cellular compartments. A general strategy is to integrate these protein maturation processes with translation, by cotranslationally engaging processing enzymes, chaperones and targeting factors with the nascent polypeptide. Precise coordination of all factors involved is critical for the efficiency and accuracy of protein synthesis and cellular homeostasis. This review provides an overview of the current knowledge on cotranslational protein maturation, with a focus on the production of cytosolic proteins in bacteria. We describe the role of the ribosome and the chaperone network in protein folding and how the dynamic interplay of all cotranslationally acting factors guides the sequence of cotranslational events. Finally, we discuss recent data demonstrating the coupling of protein synthesis with the assembly of protein complexes and end with a brief discussion of outstanding questions and emerging concepts in the field of cotranslational protein maturation.

Trigger Factor-Induced Nascent Chain Dynamics Changes Suggest Two Different Chaperone-Nascent Chain Interactions during Translation.

Protein biogenesis is poorly understood due to the ribosome that perturbs measurement attempted on the ribosome-bound nascent chain (RNC). Investigating nascent chain dynamics may provide invaluable insight into the co-translational processes such as structure formation or interaction with a chaperone [e.g., the bacterial trigger factor (TF)]. In this study, we aim to establish a platform for studying nascent chain dynamics by exploring the local environment near the fluorescent dye on site-specifically labeled RNCs with time-resolved fluorescence anisotropy. To prepare a quantitative model of fluorescence depolarization, we utilized intrinsically disordered protein bound to ribosome, which helped us couple the sub-nanosecond depolarization with the motion of the nascent chain backbone. This was consistent with zinc-finger-domain-containing RNCs, where the extent of sub-nanosecond motion decreased upon the addition of zinc when the fluorophore was in close proximity of the domain. After the characterization of disordered nascent chain dynamics, we investigated the synthesis of a model cytosolic protein, Entner-Doudoroff aldolase, labeled at different sites during various stages of translation. Depending on the stage of translation, the addition of the TF to the nascent chain led to two different responses in the nascent chain dynamics serendipitously, suggesting steric hindrance between the nascent chain and the chaperone as a mechanism for TF dissociation from the ribosome during translation. Overall, our study demonstrates the possible use of site-specific labeling and time-resolved anisotropy to gain insight on chaperone binding event at various stages of translation and hints on TF co-translational mechanism.

Nonorthogonal tRNA(cys)(Amber) for protein and nascent chain labeling.

In vitro-transcribed suppressor tRNAs are commonly used in site-specific fluorescence labeling for protein and ribosome-bound nascent chains (RNCs) studies. Here, we describe the production of nonorthogonal Bacillus subtilis tRNA(cys)(Amber) from Escherichia coli, a process that is superior to in vitro transcription in terms of yield, ease of manipulation, and tRNA stability. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNA(cys)(Amber) with lower efficiency, multiple tRNA synthetase mutants were designed to optimize aminoacylation. Aminoacylated tRNA was conjugated to a fluorophore to produce BODIPY FL-cysteinyl-tRNA(cys)(Amber), which was used to generate ribosome-bound nascent chains of different lengths with the fluorophore incorporated at various predetermined sites. This tRNA tool may be beneficial in the site-specific labeling of full-length proteins as well as RNCs for biophysical and biological research.

Diversity of chlorobiphenyl-metabolizing bacteria and their biphenyl dioxygenases in contaminated sediment.

Bacteria and bacterial communities in sites contaminated with polychlorinated biphenyls have been extensively studied in the past decades. However, there are still major gaps in the knowledge of environmental processes, especially in the behavior of previously described bacteria in vitro, their real degradation abilities and the enzymes that are involved in the degradation processes. In this work we analyzed actively degrading bacterial populations by stable isotope probing with (13)C biphenyl and (13)C-4-chlorobiphenyl as labeled substrates in the environment of sediment contaminated with polychlorinated biphenyls. We performed analysis of populations which degrade biphenyl and 4-chlorobiphenyl at concentrations similar to those of the original site. Several bacterial genera were revealed to actively participate in biphenyl and 4-chlorobiphenyl removal, some of which had not previously been described to take part in this process. We also found there are few differences in the communities metabolizing biphenyl and 4-chlorobiphenyl. Analysis of the genes responsible for substrate removal proved most of the genes to be closely related to Pseudomonas pseudoalcaligenes KF707 genes giving bacteria the ability of transforming di-para-chlorinated biphenyls.

Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription.

Here we demonstrate the use of strong anion-exchange fast performance liquid chromatography (FPLC) as a simple, fast, and robust method for RNA production by in vitro transcription. With this technique, we have purified different transcription templates from unreacted reagents in large quantities. The same buffer system could be used to readily remove nuclease contamination from the overexpressed pyrophosphatase, the important reagent for in vitro transcription. In addition, the method can be used to monitor in vitro transcription reactions to enable facile optimization of reaction conditions, and we have compared the separation performance between strong and weak anion-exchange FPLC for various transcribed RNAs, including the Diels-Alder ribozyme, the hammerhead ribozyme tRNA, and 4.5S RNA. The functionality of the purified tRNA(Cys) has been confirmed by the aminoacylation assay. Only the purification by strong anion-exchange FPLC has led to the enrichment of the functional tRNA from run-off transcripts as revealed by both enzymatic and electrophoretic analysis.

Inhibition of TDP-43 aggregation by nucleic acid binding.

The aggregation of TAR DNA-binding protein (TDP-43) has been shown as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. While evidence has suggested that mutation or truncation in TDP-43 influences its aggregation process, nevertheless, the correlation between the TDP-43 aggregation propensity and its binding substrates has not been fully established in TDP-43 proteinopathy. To address this question, we have established a platform based on the in vitro protein expression system to evaluate the solubility change of TDP-43 in response to factors such as nucleotide binding and temperature. Our results suggest that the solubility of TDP-43 is largely influenced by its cognate single-strand DNA (ssDNA) or RNA (ssRNA) rather than hnRNP, which is known to associate with TDP-43 C-terminus. The direct interaction between the refolded TDP-43, purified from E.coli, and ssDNA were further characterized by Circular Dichroism (CD) as well as turbidity and filter binding assay. In addition, ssDNA or ssRNA failed to prevent the aggregation of the F147L/F149L double mutant or truncated TDP-43 (TDP208-414). Consistently, these two mutants form aggregates, in contrast with the wild-type TDP-43, when expressed in Neuro2a cells. Our results demonstrate an intimate relationship between the solubility of TDP-43 and its DNA or RNA binding affinity, which may shed light on the role of TDP-43 in ALS and FTLD.

Plant secondary metabolite-induced shifts in bacterial community structure and degradative ability in contaminated soil.

The aim of the study was to investigate how selected natural compounds (naringin, caffeic acid, and limonene) induce shifts in both bacterial community structure and degradative activity in long-term polychlorinated biphenyl (PCB)-contaminated soil and how these changes correlate with changes in chlorobiphenyl degradation capacity. In order to address this issue, we have integrated analytical methods of determining PCB degradation with pyrosequencing of 16S rRNA gene tag-encoded amplicons and DNA-stable isotope probing (SIP). Our model system was set in laboratory microcosms with PCB-contaminated soil, which was enriched for 8 weeks with the suspensions of flavonoid naringin, terpene limonene, and phenolic caffeic acid. Our results show that application of selected plant secondary metabolites resulted in bacterial community structure far different from the control one (no natural compound amendment). The community in soil treated with caffeic acid is almost solely represented by Proteobacteria, Acidobacteria, and Verrucomicrobia (together over 99 %). Treatment with naringin resulted in an enrichment of Firmicutes to the exclusion of Acidobacteria and Verrucomicrobia. SIP was applied in order to identify populations actively participating in 4-chlorobiphenyl catabolism. We observed that naringin and limonene in soil foster mainly populations of Hydrogenophaga spp., caffeic acid Burkholderia spp. and Pseudoxanthomonas spp. None of these populations were detected among 4-chlorobiphenyl utilizers in non-amended soil. Similarly, the degradation of individual PCB congeners was influenced by the addition of different plant compounds. Residual content of PCBs was lowest after treating the soil with naringin. Addition of caffeic acid resulted in comparable decrease of total PCBs with non-amended soil; however, higher substituted congeners were more degraded after caffeic acid treatment compared to all other treatments. Finally, it appears that plant secondary metabolites have a strong effect on the bacterial community structure, activity, and associated degradative ability.

Cotranslational protein folding within the ribosome tunnel influences trigger-factor recruitment.

In recent years, various folding zones within the ribosome tunnel have been identified and explored through x-ray, cryo-electron microscopy (cryo-EM), and molecular biology studies. Here, we generated ribosome-bound nascent polypeptide complexes (RNCs) with different polyalanine (poly-A) inserts or signal peptides from membrane/secretory proteins to explore the influence of nascent chain compaction in the Escherichia coli ribosome tunnel on chaperone recruitment. By employing time-resolved fluorescence resonance energy transfer and immunoblotting, we were able to show that the poly-A inserts embedded in the passage tunnel can form a compacted structure (presumably helix) and reduce the recruitment of Trigger Factor (TF) when the helical motif is located in the region near the tunnel exit. Similar experiments on nascent chains containing signal sequences that may form compacted structural motifs within the ribosome tunnel and lure the signal recognition particle (SRP) to the ribosome, provided additional evidence that short, compacted nascent chains interfere with TF binding. These findings shed light on the possible controlling mechanism of nascent chains within the tunnel that leads to chaperone recruitment, as well as the function of L23, the ribosomal protein that serves as docking sites for both TF and SRP, in cotranslational protein targeting.