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Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome  [PDF]
Surojit Mondal, Bani Kumar Pathak, Sutapa Ray, Chandana Barat
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0101293
Abstract: Background The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. Results The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. Conclusion Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.
The Circadian Clock Coordinates Ribosome Biogenesis  [PDF]
Céline Jouffe equal contributor,Gaspard Cretenet equal contributor,Laura Symul,Eva Martin,Florian Atger,Felix Naef,Frédéric Gachon
PLOS Biology , 2013, DOI: 10.1371/journal.pbio.1001455
Abstract: Biological rhythms play a fundamental role in the physiology and behavior of most living organisms. Rhythmic circadian expression of clock-controlled genes is orchestrated by a molecular clock that relies on interconnected negative feedback loops of transcription regulators. Here we show that the circadian clock exerts its function also through the regulation of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator directly regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis.
Evolution of the holozoan ribosome biogenesis regulon
Seth J Brown, Michael D Cole, Albert J Erives
BMC Genomics , 2008, DOI: 10.1186/1471-2164-9-442
Abstract: Here we present genome-wide analyses indicating that a distinct mode of RiBi regulation co-evolved with the E(CG)-binding, Myc:Max bHLH heterodimer complex in a stem-holozoan, the ancestor of both Metazoa and Choanoflagellata, the protozoan group most closely related to animals. These results show that this mode of regulation, characterized by an E(CG)-bearing core-promoter, is specific to almost all of the known genes involved in ribosome biogenesis in these genomes. Interestingly, this holozoan RiBi promoter signature is absent in nematode genomes, which have not only secondarily lost Myc but are marked by invariant cell lineages typically producing small body plans of 1000 somatic cells. Furthermore, a detailed analysis of 10 fungal genomes shows that this holozoan signature in RiBi genes is not found in hemiascomycete fungi, which evolved their own unique regulatory signature for the RiBi regulon.These results indicate that a Myc regulon, which is activated in proliferating cells during normal development as well as during tumor progression, has primordial roots in the evolution of an inducible growth regime in a protozoan ancestor of animals. Furthermore, by comparing divergent bHLH repertoires, we conclude that regulation by Myc but not by other bHLH genes is responsible for the evolutionary maintenance of E(CG) sites across the RiBi suite of genes.Ribosome biogenesis (RiBi) is a primary function of the nucleolus [1-3]. In the nucleolus, rRNA molecules are synthesized as precursors by DNA-directed RNA Pol I and Pol III. Nascent rRNAs then undergo extensive chemical modifications and RNA cleavage reactions. Numerous RiBi proteins are involved both in this enzymatic processing as well in assisting with proper rRNA folding to produce functional ribosomal subunits [1]. Synthesizing functional ribosomes requires immense coordination because gene products from all three DNA-dependent RNA polymerases are required to ensure proper stoichiometry of ribosomal components
tRNA-Derived Fragments Target the Ribosome and Function as Regulatory Non-Coding RNA in Haloferax volcanii  [PDF]
Jennifer Gebetsberger,Marek Zywicki,Andrea Künzi,Norbert Polacek
Archaea , 2012, DOI: 10.1155/2012/260909
Abstract: Nonprotein coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. These RNAs either originate from their individual transcription units or are processing products from longer precursor RNAs. For example, tRNA-derived fragments (tRFs) have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNA candidates. Here we present evidence that tRFs from the halophilic archaeon Haloferax volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome, a 26-residue-long fragment originating from the 5′ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine tuning the rate of protein production. 1. Introduction Recent research revealed small nonprotein coding RNAs (ncRNAs) as pivotal players in regulatory networks shaping cellular life in all three phylogenetic domains (reviewed in [1]). Regulatory functions of ncRNAs are diverse ranging from chromosome biology, to epigenetics, transcription, and translation regulation [2]. This variety of ncRNA functions is mirrored by their complex genomics and biogenesis. Recent data suggest that a single ncRNA transcript can adopt different structures and thus perform distinct functional roles depending on different posttranscriptional processing events. Four years ago it could be shown that a functional small nucleolar RNA, which was initially processed from an mRNA intron, could function as a microRNA after further processing took place [3]. Many other reports followed expanding the list of ncRNA species that are target of further downstream processing into novel regulatory entities [4–7] thus it appears that we have just started to disentangle the hidden layers of the transcriptome. One recent example of ncRNA processing presents the emerging group of RNA fragments derived from mature tRNAs or precursor tRNAs [8]. These tRNA-derived fragments (tRFs) have long been regarded as random byproducts of tRNA biogenesis or degradation, but are now recognized as emerging players in tRNA biology. tRFs have been
Connecting the Kinetics and Energy Landscape of tRNA Translocation on the Ribosome  [PDF]
Paul C. Whitford ,Scott C. Blanchard,Jamie H. D. Cate,Karissa Y. Sanbonmatsu
PLOS Computational Biology , 2013, DOI: 10.1371/journal.pcbi.1003003
Abstract: Functional rearrangements in biomolecular assemblies result from diffusion across an underlying energy landscape. While bulk kinetic measurements rely on discrete state-like approximations to the energy landscape, single-molecule methods can project the free energy onto specific coordinates. With measures of the diffusion, one may establish a quantitative bridge between state-like kinetic measurements and the continuous energy landscape. We used an all-atom molecular dynamics simulation of the 70S ribosome (2.1 million atoms; 1.3 microseconds) to provide this bridge for specific conformational events associated with the process of tRNA translocation. Starting from a pre-translocation configuration, we identified sets of residues that collectively undergo rotary rearrangements implicated in ribosome function. Estimates of the diffusion coefficients along these collective coordinates for translocation were then used to interconvert between experimental rates and measures of the energy landscape. This analysis, in conjunction with previously reported experimental rates of translocation, provides an upper-bound estimate of the free-energy barriers associated with translocation. While this analysis was performed for a particular kinetic scheme of translocation, the quantitative framework is general and may be applied to energetic and kinetic descriptions that include any number of intermediates and transition states.
Nom1 Mediates Pancreas Development by Regulating Ribosome Biogenesis in Zebrafish  [PDF]
Wei Qin, Zelin Chen, Yihan Zhang, Ruibin Yan, Guanrong Yan, Song Li, Hanbing Zhong, Shuo Lin
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0100796
Abstract: Ribosome biogenesis is an important biological process for proper cellular function and development. Defects leading to improper ribosome biogenesis can cause diseases such as Diamond-Blackfan anemia and Shwachman-Bodian-Diamond syndrome. Nucleolar proteins are a large family of proteins and are involved in many cellular processes, including the regulation of ribosome biogenesis. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish line carrying mutation in nucleolar protein with MIF4G domain 1 (nom1), which encodes a conserved nulceolar protein with a role in pre-rRNA processing. Zebrafish nom1 mutants exhibit major defects in endoderm development, especially in exocrine pancreas. Further studies revealed that impaired proliferation of ptf1a-expressing pancreatic progenitor cells mainly contributed to the phenotype. RNA-seq and molecular analysis showed that ribosome biogenesis and pre-mRNA splicing were both affected in the mutant embryos. Several defects of ribosome assembly have been shown to have a p53-dependent mechanism. In the nom1 mutant, loss of p53 did not rescue the pancreatic defect, suggesting a p53-independent role. Further studies indicate that protein phosphatase 1 alpha, an interacting protein to Nom1, could partially rescue the pancreatic defect in nom1 morphants if a human nucleolar localization signal sequence was artificially added. This suggests that targeting Pp1α into the nucleolus by Nom1 is important for pancreatic proliferation. Altogether, our studies revealed a new mechanism involving Nom1 in controlling vertebrate exocrine pancreas formation.
Inhibition of Human Dyskerin as a New Approach to Target Ribosome Biogenesis  [PDF]
Laura Rocchi, Arménio J. M. Barbosa, Carmine Onofrillo, Alberto Del Rio, Lorenzo Montanaro
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0101971
Abstract: The product of the DKC1 gene, dyskerin, is required for both ribosome biogenesis and telomerase complex stabilization. Targeting these cellular processes has been explored for the development of drugs to selectively or preferentially kill cancer cells. Presently, intense research is conducted involving the identification of new biological targets whose modulation may simultaneously interfere with multiple cellular functions that are known to be hyper-activated by neoplastic transformations. Here, we report, for the first time, the computational identification of small molecules able to inhibit dyskerin catalytic activity. Different in silico techniques were applied to select compounds and analyze the binding modes and the interaction patterns of ligands in the human dyskerin catalytic site. We also describe a newly developed and optimized fast real-time PCR assay that was used to detect dyskerin pseudouridylation activity in vitro. The identification of new dyskerin inhibitors constitutes the first proof of principle that the pseudouridylation activity can be modulated by means of small molecule agents. Therefore, the presented results, obtained through the usage of computational tools and experimental validation, indicate an alternative therapeutic strategy to target ribosome biogenesis pathway.
Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics  [PDF]
Zhihua Li,Insuk Lee,Emily Moradi,Nai-Jung Hung,Arlen W. Johnson,Edward M. Marcotte
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1000213
Abstract: Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics—an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies—to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes—most with human orthologs—to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.
Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics  [PDF]
Zhihua Li,Insuk Lee,Emily Moradi,Nai-Jung Hung,Arlen W. Johnson ,Edward M. Marcotte
PLOS Biology , 2009, DOI: 10.1371/journal.pbio.1000213
Abstract: Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics—an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies—to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes—most with human orthologs—to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.
Staphylococcus aureus and Escherichia coli have disparate dependences on KsgA for growth and ribosome biogenesis  [cached]
O’Farrell Heather C,Rife Jason P
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-244
Abstract: Background The KsgA methyltransferase has been conserved throughout evolution, methylating two adenosines in the small subunit rRNA in all three domains of life as well as in eukaryotic organelles that contain ribosomes. Understanding of KsgA’s important role in ribosome biogenesis has been recently expanded in Escherichia coli; these studies help explain why KsgA is so highly conserved and also suggest KsgA’s potential as an antimicrobial drug target. Results We have analyzed KsgA’s contribution to ribosome biogenesis and cell growth in Staphylococcus aureus. We found that deletion of ksgA in S. aureus led to a cold-sensitive growth phenotype, although KsgA was not as critical for ribosome biogenesis as it was shown to be in E. coli. Additionally, the ksgA knockout strain showed an increased sensitivity to aminoglycoside antibiotics. Overexpression of a catalytically inactive KsgA mutant was deleterious in the knockout strain but not the wild-type strain; this negative phenotype disappeared at low temperature. Conclusions This work extends the study of KsgA, allowing comparison of this aspect of ribosome biogenesis between a Gram-negative and a Gram-positive organism. Our results in S. aureus are in contrast to results previously described in E. coli, where the catalytically inactive protein showed a negative phenotype in the presence or absence of endogenous KsgA.
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