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1.
Annu Rev Microbiol ; 76: 193-210, 2022 09 08.
Article in English | MEDLINE | ID: mdl-35609945

ABSTRACT

The pace and efficiency of ribosomal subunit production directly impact the fitness of bacteria. Biogenesis demands more than just the union of ribosomal components, including RNA and proteins, to form this functional ribonucleoprotein particle. Extra-ribosomal protein factors play a fundamental role in the efficiency and efficacy of ribosomal subunit biogenesis. A paucity of data on intermediate steps, multiple and overlapping pathways, and the puzzling number of functions that extra-ribosomal proteins appear to play in vivo make unraveling the formation of this macromolecular assemblage difficult. In this review, we outline with examples the multinodal landscape of factor-assisted mechanisms that influence ribosome synthesis in bacteria. We discuss in detail late-stage events that mediate correct ribosome formation and the transition to translation initiation and thereby ensure high-fidelity protein synthesis.


Subject(s)
Ribosomal Proteins , Ribosomes , Bacteria/genetics , Bacteria/metabolism , Protein Biosynthesis , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomes/metabolism
2.
Biochimie ; 191: 104-117, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34508826

ABSTRACT

The bacterial ribosomal protein S15 is located in the platform, a functional region of the 30S ribosomal subunit. While S15 is critical for in vitro formation of E. coli small subunits (SSUs), it is dispensable for in vivo biogenesis and growth. In this work, a novel synergistic interaction between rpsO, the gene that encodes S15, and rnc (the gene that encodes RNase III), was uncovered in E. coli. RNase III catalyzes processing of precursor ribosomal RNA (rRNA) transcripts and thus is involved in functional ribosome subunit maturation. Strains lacking S15 (ΔrpsO), RNase III (Δrnc) or both genes were examined to understand the relationship between these two factors and the impact of this double deletion on rRNA processing and SSU maturation. The double deletion of rpsO and rnc partially alleviates the observed cold sensitivity of ΔrpsO alone. A novel 16S rRNA precursor (17S∗ rRNA) that is detected in free 30S subunits of Δrnc is incorporated in 70S-like ribosomes in the double deletion. The stable accumulation of 17S∗ rRNA suggests that timing of processing events is closely coupled with SSU formation events in vivo. The double deletion has a suppressive effect on the cell elongation phenotype of ΔrpsO. The alteration of the phenotypes associated with S15 loss, due to the absence of RNase III, indicates that pre-rRNA processing and improvement of growth, relative to that observed for ΔrpsO, are connected. The characterization of the functional link between the two factors illustrates that there are redundancies and compensatory pathways for SSU maturation.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Ribonuclease III/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Ribonuclease III/genetics , Ribosomal Proteins/genetics , Ribosomes/genetics
3.
Article in English | MEDLINE | ID: mdl-33294101

ABSTRACT

A large body of data suggests that implementing active learning practices in a STEM classroom contributes to increased success in both achievement of student learning outcomes and retention of students. Despite these findings, significant barriers exist for instructors implementing active learning strategies in their undergraduate classrooms. These barriers can be effectively addressed by providing sustained support to instructors and postdoctoral trainees interested in implementing active learning strategies in their teaching practice. The Promoting Active Learning and Mentoring (PALM) network attains this objective by connecting instructors interested in learning more about active learning (Fellows) with individuals who have extensive expertise related to this practice (mentors). These facilitated connections occur in the form of active mentorship for a year or more, virtual journal clubs, and biannual gatherings of PALM Fellows and mentors. Here, we describe the foundation on which PALM was built and explain how a successful mentorship program can pave the way for educators to adapt and implement evidence-based practices like active learning in a college classroom.

4.
PLoS One ; 10(4): e0120060, 2015.
Article in English | MEDLINE | ID: mdl-25879934

ABSTRACT

The loss of the large ribosomal protein L9 causes a reduction in translation fidelity by an unknown mechanism. To identify pathways affected by L9, we identified mutants of E. coli that require L9 for fitness. In a prior study, we characterized L9-dependent mutations in the essential GTPase Der (EngA). Here, we describe a second class of L9-dependent mutations that either compromise or inactivate elongation factor P (EF-P, eIF5A in eukaryotes). Without L9, Δefp cells are practically inviable. Cell fractionation studies revealed that, in both the Der and EF-P mutant cases, L9's activity reduces immature 16S rRNA in 30S particles and partially restores the abundance of monosomes. Inspired by these findings, we discovered that L9 also enhances 16S maturation in wild-type cells. Surprisingly, although the amount of immature 16S in 30S particles was found to be elevated in ΔrplI cells, the amount in polysomes was low and inversely correlated with the immature 16S abundance. These findings provide an explanation for the observed fitness increases afforded by L9 in these mutants and reveal particular physiological conditions in which L9 becomes critical. Additionally, L9 may affect the partitioning of small subunits containing immature 16S rRNA.


Subject(s)
Peptide Elongation Factors/genetics , Ribosomal Proteins/physiology , Mutation , RNA, Ribosomal, 16S/metabolism
5.
J Bacteriol ; 195(16): 3682-91, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23772068

ABSTRACT

Ribosomal protein L9 is a component of all eubacterial ribosomes, yet deletion strains display only subtle growth defects. Although L9 has been implicated in helping ribosomes maintain translation reading frame and in regulating translation bypass, no portion of the ribosome-bound protein seems capable of contacting either the peptidyltransferase center or the decoding center, so it is a mystery how L9 can influence these important processes. To reveal the physiological roles of L9 that have maintained it in evolution, we identified mutants of Escherichia coli that depend on L9 for fitness. In this report, we describe a class of L9-dependent mutants in the ribosome biogenesis GTPase Der (EngA/YphC). Purified mutant proteins were severely compromised in their GTPase activities, despite the fact that the mutations are not present in GTP hydrolysis sites. Moreover, although L9 and YihI complemented the slow-growth der phenotypes, neither factor could rescue the GTPase activities in vitro. Complementation studies revealed that the N-terminal domain of L9 is necessary and sufficient to improve the fitness of these Der mutants, suggesting that this domain may help stabilize compromised ribosomes that accumulate when Der is defective. Finally, we employed a targeted degradation system to rapidly deplete L9 from a highly compromised der mutant strain and show that the L9-dependent phenotype coincides with a cell division defect.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , GTP-Binding Proteins/metabolism , Ribosomal Proteins/metabolism , Escherichia coli/cytology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , GTP-Binding Proteins/genetics , Gene Expression Regulation, Bacterial/physiology , Models, Molecular , Mutation , Protein Conformation , Ribosomal Proteins/genetics
6.
Cancer Res ; 70(1): 418-27, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-20028870

ABSTRACT

Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM.


Subject(s)
Brain Neoplasms/metabolism , Glioblastoma/metabolism , Neoplasm Invasiveness/pathology , Receptor, Notch1/metabolism , TRPC Cation Channels/metabolism , Adult , Blotting, Western , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Hypoxia/physiology , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Immunohistochemistry , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Neoplasm Invasiveness/genetics , RNA, Small Interfering , Receptor, Notch1/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , TRPC Cation Channels/genetics , TRPC6 Cation Channel
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