No description available.

<p>While much is known about the abundance and genetic diversity of environmental microbial communities, little is known about their taxon-specific activity. In this thesis I address this gap using a model marine microbe, the cyanobacterium <italic>Prochlorococcus spp.</italic>, which numerically dominates tropical and subtropical open oceans and encompasses a group of genetically defined clades that are ecologically distinct. Ribosomal RNA is a promising indicator of in situ activity because of its essential role in protein synthesis as well as its phylogenetic information, which could be used to distinguish clades among mixed populations. Here I show that, in a laboratory system the specific growth rate of representative <italic>Prochlorococcus</italic> strains could be quantitative predicted from cellular rRNA content (assessed by RT-qPCR), cell size (assessed by flow cytometry) and temperature. Applying this approach in the field, I show unique clade-specific activity patterns for <italic>Prochlorococcus</italic>. For example, vertically within the euphotic zone, eHL-II activity is strongly impacted by light and is consistent with patterns of photosynthesis and on a horizontal transect from Hawaii to San Diego, eHL-I and eHL-II activities exhibit significant transitions and appear to be regulated by temperature, nutrient and vertical mixing gradients. Using ribosomal tag pyrosequencing of DNA and RNA, I have extended our observation to the Eubacterial community and described the biomass distribution (rDNA) and activity (rRNA) patterns from two representative depths (25 and 100 m) at a well-studied oligotrophic ocean station. These results show that for some populations the abundances and activities are significantly uncoupled, which suggests substantial top-down controls or physical transport processes. Further exploring the taxon-specific activity patterns along with abundances and environmental variables across time and space is essential to better understanding the dynamics of a complex microbial system as well as predicting the consequences of environmental change.</p> / Dissertation

The bacterial ribosome is essential to cell growth yet little is known about how its proteins attain their mature structures. Recent studies indicate that certain Staphlyococcus aureus bacteriophage protein sequences contain specific sites that may be cleaved by a non-bacteriophage enzyme (Poliakov et al. 2008). The phage cleavage site was found to bear sequence similarity to the N-terminus of S. aureus ribosomal protein L27. Previous studies in E. coli (Wower et al.1998; Maguire et al. 2005) found that L27 is situated adjacent to the ribosomal peptidyl transferase site, where it likely aids in new peptide formation. The predicted S. aureus L27 protein contains an additional N-terminal sequence not observed within the N-terminus of the otherwise similar E. coli L27; this sequence appears to be cleaved, indicating yet-unobserved ribosomal protein post-translational processing and use of host processes by phage. Phylogenetic analysis shows that L27 processing has the potential to be highly conserved. Further study of this phenomenon may aid antibiotic development.

Thesis advisor: Michelle M. Meyer / Bacteria commonly utilize cis-acting mRNA structures that bind specific molecules to control gene expression in response to changing cellular conditions. Examples of these ligand-sensing RNA cis-regulators are found throughout the bacterial world and include riboswitches, which interact with small metabolites to modulate the expression of fundamental metabolic genes, and the RNA structures that bind select ribosomal proteins to regulate entire ribosomal protein operons. Despite advances in both non-coding RNA discovery and validation, many predicted regulatory RNA motifs remain uncharacterized and little work has examined how RNA cis-regulators behave within their physiological context in the cell. Furthermore, it is not well understood how structured RNA regulators emerge and are maintained within bacterial genomes. In this thesis, I validate the biological function of a conserved RNA cis-regulator of ribosomal protein synthesis previously discovered by my group using bioinformatic approaches. I then investigate how bacteria respond to the loss of two different cis-regulatory RNA structures. Using Bacillus subtilis as a model organism, I introduce point mutations into the native loci of the ribosomal protein L20-interacting RNA cis-regulator and the tandem glycine riboswitch and assay the strains for fitness defects. I find that disrupting these regulatory RNA structures results in severe mutant phenotypes, especially under harsh conditions such as low temperatures or high glycine concentrations. Together, this body of work highlights the advantages of examining RNA behavior within its biological context and emphasizes the important role RNA cis-regulators play in overall organismal viability. My studies shed light on the selective pressures that impact structured RNA evolution in vivo and reinforce the potential of cis-regulatory RNAs as novel antimicrobial targets. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A biogênese do ribossomo é um processo complexo, altamente ordenado e regulado, no qual o transcrito primário é processado por endo e exonucleases para gerar os RNAs ribossomais maduros. Este processo foi melhor caracterizado em Saccharomyces cerevisiae, porém alguns fatores atuantes em humanos tiveram uma função divergente descrita. Um desses fatores é a proteína NIP7, altamente conservada em eucariotos, que atua na formação da subunidade ribossomal 60S, em levedura, e 40S, em humanos. Assim, esse trabalho propôs a caracterização funcional da proteína CrNIP7, homóloga a NIP7, presente em Chlamydomonas reinhardtii. C. reinhardtii é uma alga verde unicelular ancestral a plantas, utilizada como modelo eucarioto para estudos de fotossíntese e de flagelos. Nesse trabalho, um estudo de complementação funcional foi realizado utilizando duas linhagens de Saccharomyces cerevisiae diferentes e em ambas CrNIP7 complementou a função de Nip7p de leveduras, indicando uma participação na síntese da subunidade 60S do ribossomo. Uma busca por parceiros de interação de CrNIP7 foi também realizada, utilizando CrNIP7 como isca para rastrear uma biblioteca de cDNA de C. reinhardtii em sistema de duplo híbrido em leveduras, o que resultou em dois novos potenciais parceiros de interação. Esses parceiros foram identificados como proteínas preditas conceitualmente no genoma de C. reinhardtii, denominadas Predicted e G-patch. Adicionalmente, a interação entre CrNIP7 e CrSBDS, proteína homóloga a Sdo1 (de levedura) e HsSBDS (de humanos), foi confirmada através de um experimento de duplo híbrido dirigido. A interação entre as proteínas CrNIP7 e CrSBDS foi validada por pull down e um teste preliminar sugeriu que CrNIP7 e Predicted também interagem in vitro. Análises de bioinformática indicam que Predicted, G-patch e CrSBDS tenham regiões intrinsicamente desordenadas, as quais podem se estruturar na interação com seus parceiros. Em conjunto, os resultados desse trabalho contribuem para entendimento do papel de CrNIP7 na biogênese de ribossomos em Chlamydomonas reinhardtii em comparação com outros modelos eucarióticos. / Ribosome biogenesis is a complex, highly regulated and ordered process in which the primary transcript is processed by endo- and exonucleases to generate the mature ribosomal RNAs. This process was best characterized in Saccharomyces cerevisiae, but some factors have been described in humans with different function. One of these divergent factors is NIP7, a highly conserved protein in eukaryotes, which acts in the formation of ribosomal 60S subunit, in yeast, and 40S, in humans. Based on this, this work proposed the functional characterization of CrNIP7 protein, homologous to NIP7, from Chlamydomonas reinhardtii. C. reinhardtii is a green alga, ancestral to plants, that is used as an eukaryote model for photosynthesis and flagella studies. In this study, a functional complementation assay was performed using two different strains of Saccharomyces cerevisiae and, in both approaches, CrNIP7 protein complemented the function of Nip7p from yeast, indicating its participation in the synthesis of the 60S ribosomal subunit. A two-hybrid assay was carried out using CrNIP7 as bait to screen a C. reinhardtii cDNA library in order to find out CrNIP7 interaction partners, wich resulted in two novel potentially partners. The interacting proteins were identified as conceptually predicted proteins in the genome of C. reinhardtii and were called Predicted and G-patch. Additionally, the interaction between CrNIP7 and CrSBDS, a protein homologous to Sdo1 (yeast) and HsSBDS (humans), was confirmed by a direct two-hybrid assay. The interaction between CrNIP7 and CrSBDS proteins was validated by pull down and a preliminary test suggested that CrNIP7 and Predicted also interact in vitro. Bioinformatics analyzes indicate that Predicted, G-patch and CrSBDS have intrinsically disordered regions, which can be ordered in the moment of interaction. Taken together, the results of this work contribute to understand the role played by CrNIP7 in ribosome biogenesis in Chlamydomonas reinhardtii compared to other eukaryotic models.

The eukaryotic ribosome is a large molecular machine consisting of ~80 ribosomal proteins and 4 rRNAs. The 40S and 60S ribosomal subunits are assembled in the nucleolus by ~200 helper proteins then shipped into the cytoplasm or to the endoplasmic reticulum where protein translation takes place. Eventually ribosomes are removed from the cytoplasm and recycled through ribophagy, however, very is little is known about ribosomal protein exchange after assembly but before ribophagy. Using kinetic turnover measurements of ribosomal proteins and rRNA in vivo we determined ribosomal protein replacement rates are diverse suggesting ribosomal components may be replaced without destruction of the entire ribosome. Measurements from ad libitum fed and dietary restricted mice provide strong evidence that RPL10 exchanges rates are dramatically different between AL and DR while synthesis and degradation do not change. RPL10 turnover can be described using a two-pool kinetic model, which may be applied to many ribosomal proteins.

Protein synthesis is a fundamental and ultimate process in living cells. Cells possess sophisticated machineries and continuously carry out complex processes. Monitoring protein synthesis in living cells not only inform us about the mechanism of translation but also deepen our insights about all aspects of life. Understanding the structure and mechanism of the ribosome and its associated factors helped us enlarge our knowledge on protein synthesis. Recently, with the dramatic advances of high-throughput sequencing and bioinformatics, a new technique called ribosome profiling emerged. By retrieving mRNA fragments protected by translating ribosomes, ribosome profiling reveals global ribosome occupancy along mRNAs in living cells, which can inform us with the identity and quantity of proteins being made. Easily adapted to other organisms, ribosome profiling technique is expanding its application in revealing various cellular activities as well as the knowledge on protein synthesis. Here, we report the mechanism of translating mRNA cleavage by endoribonuclease RelE in vivo. RelE is an endoribonuclease that is induced during nutrient deficiency stress and specifically cleaves translating mRNAs upon binding to the ribosomal A site. Overexpression of RelE in living cells causes growth arrest by inhibiting global translation. We monitored RelE activity in vivo upon overexpression using ribosome profiling. The data show that RelE actively cuts translating mRNAs whenever the ribosomal A site is accessible, resulting in truncated mRNAs. RelE causes the ribosome complexes to accumulate near the 5' end of genes as the process of ribosome rescue, translation, and cleavage by RelE repeats. RelE cleavage specific sub-codon level ribosome profiling data also represent reading frame in Escherichia coli and sequence specificity of RelE cleavage in vivo. We report another ribosome profiling study on a methyltransferase TrmD in E. coli. TrmD is known to methylate G37 (the residue at 3' side of anticodon) of some tRNAs and be responsible for codon-anticodon interaction. We constructed a TrmD depletion E. coli strain, whose deletion results in lethality of cells. Resulting depletion of m1G37 in the strain leads to growth arrest. Lack of m1G37 of some tRNAs whose codons start with C showed frequent frameshift when translating the gene message in vitro. By using ribosome profiling, we successfully observed significant difference on translation process when codons interact with anticodons of tRNAs lacking m1G37. The data reveal slow translation rate or pauses on the tRNAs when missing the appropriate methylation, which corresponds to the previous biochemical data in vitro.

No description available.

<p>The aim of this work was to understand the molecular mechanism of translation and the mechanism of translation termination, in particular. Cleavage of peptidyl-tRNA and peptide release terminates translation of mRNA on the ribosome. In prokaryotes, three release factors (RFs) are involved in this process. RF1 and RF2 recognise the three stop codons on mRNA and induce hydrolysis of the ester bond in peptidyl-tRNA. RF3 accelerates the rate of RF1 and RF2 recycling between ribosome in a GTP-dependent manner. We have clarified the mechanism of action of peptide release factor RF3. In the cell, free RF3 is in the GDP conformation. When RF3∙GDP binds to ribosome in complex with RF1 or RF2, these ribosome complexes act as guanine exchange factors for RF3 by inducing rapid dissociation of GDP. If, and only if, the peptide has been removed from tRNA, GDP is quickly replaced by GTP. Binding of GTP to RF3 induces a conformation of the factor with high affinity for the ribosome, which forces RF1 or RF2 to rapidly dissociate. Subsequent hydrolysis of GTP on RF3 induces a factor conformation with low affinity for the ribosome and rapid release of RF3∙GDP. It was further shown how the position of peptidyl-tRNA on the ribosome and the presence or absence of its peptide regulates the binding and GTPase activity of translation factors IF2, EF-G and EF-Tu. The result explains how idling GTPase hydrolysis and negative interference between different translation factors are minimized in living cells. The present biochemical observations, in conjunction with cryo-EM results, lead to new proposals for the role of hybrid sites in translocation of tRNAs, recycling of RF1 and RF2 by RF3 and recycling of post-termination ribosomes back to a new round of initiation.</p>

No description available.