Involvement of a region of 23S ribosomal RNA of {\it E. coli\/} in decoding of termination codons

Involvement of E. coli 23S ribosomal RNA (rRNA) in decoding of termination codons was first indicated by the characterization of a 23S rRNA mutant that causes UGA-specific nonsense suppression. The work described here was begun to test the hypothesis that more 23S rRNA suppressors of specific nonsense mutations can be isolated and that they would occur non-randomly in the rRNA genes and be clustered in specific, functionally significant regions of rRNA.^ Approximately 2 kilobases of the gene for 23S rRNA were subjected to PCR random mutagenesis and the amplified products screened for suppression of nonsense mutations in trpA. All of the suppressor mutations obtained were located in a thirty-nucleotide part of the GTPase center, a conserved rRNA sequence and structure, and they and others made in that region by site-directed mutagenesis were shown to be UGA-specific in their suppression of termination codon mutations. These results proved the initial hypothesis and demonstrated that a group of nucleotides in this region are involved in decoding of the UGA termination codon. Further, it was shown that limitation of cellular availability or synthesis of L11, a ribosomal protein that binds to the GTPase center rRNA, resulted in suppression of termination codon mutations, suggesting the direct involvement of L11 in termination in vivo.^ Finally, in vivo analysis of certain site-specific mutations made in the GTPase center RNA demonstrated that (a) the G$\cdot$A base pair closing the hexanucleotide hairpin loop was not essential for normal termination, (b) the "U-turn" structure in the 1093 to 1098 hexaloop is critical for normal termination, (c) nucleotides A1095 and A1067, necessary for the binding to ribosomes of thiostrepton, an antibiotic that inhibits polypeptide release factor binding to ribosomes in vitro, are also necessary for normal peptide chain termination in vivo, and (d) involvement of this region of rRNA in termination is determined by some unique subset structure that includes particular nucleotides rather than merely by a general structural feature of the GTPase center.^ This work advances the understanding of peptide chain termination by demonstrating that the GTPase region of 23S rRNA participates in recognition of termination codons, through an associated ribosomal protein and specific conserved nucleotides and structural motifs in its RNA. ^

Functions and intramolecular interactions of ribosomal RNA in decoding of termination codons

Two regions in the 3$\prime$ domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3$\prime$ domain of 16S rRNA. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.^ With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484$\Delta$) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The $\Delta$2484 and A1093/$\Delta$2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.^ Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned the genes for EF-G and all three release factors into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay. (Abstract shortened by UMI.) ^

The sequence comparison index: A novel method for comparing proteins and proteomes

Historically morphological features were used as the primary means to classify organisms. However, the age of molecular genetics has allowed us to approach this field from the perspective of the organism's genetic code. Early work used highly conserved sequences, such as ribosomal RNA. The increasing number of complete genomes in the public data repositories provides the opportunity to look not only at a single gene, but at organisms' entire parts list. ^ Here the Sequence Comparison Index (SCI) and the Organism Comparison Index (OCI), algorithms and methods to compare proteins and proteomes, are presented. The complete proteomes of 104 sequenced organisms were compared. Over 280 million full Smith-Waterman alignments were performed on sequence pairs which had a reasonable expectation of being related. From these alignments a whole proteome phylogenetic tree was constructed. This method was also used to compare the small subunit (SSU) rRNA from each organism and a tree constructed from these results. The SSU rRNA tree by the SCI/OCI method looks very much like accepted SSU rRNA trees from sources such as the Ribosomal Database Project, thus validating the method. The SCI/OCI proteome tree showed a number of small but significant differences when compared to the SSU rRNA tree and proteome trees constructed by other methods. Horizontal gene transfer does not appear to affect the SCI/OCI trees until the transferred genes make up a large portion of the proteome. ^ As part of this work, the Database of Related Local Alignments (DaRLA) was created and contains over 81 million rows of sequence alignment information. DaRLA, while primarily used to build the whole proteome trees, can also be applied shared gene content analysis, gene order analysis, and creating individual protein trees. ^ Finally, the standard BLAST method for analyzing shared gene content was compared to the SCI method using 4 spirochetes. The SCI system performed flawlessly, finding all proteins from one organism against itself and finding all the ribosomal proteins between organisms. The BLAST system missed some proteins from its respective organism and failed to detect small ribosomal proteins between organisms. ^

Control of chromosomal rearrangements in {\it Escherichia coli\/}

A complete physical map of Escherichia coli K-12 strain MG1655 was constructed by digesting chromosomal DNA with the infrequently cutting restriction enzymes NotI, SfiI and XbaI and separating the fragments by pulsed field gel electrophoresis. The map was used to compare six K-12 strains of E. coli. Although several differences were noted and localized, the map of MG1655 was representative of all the K-12 strains tested. The maps were also used to analyze chromosomal rearrangements in the E. coli strain MG1655. The spontaneous and UV induced frequencies of tandem duplication formation were measured at several loci distributed around the chromosome. The spontaneous duplication frequency varied from 10$\sp{-5}$ to 10$\sp{-3}$ and increased at least ten-fold following mild UV irradiation treatment. Duplications of several regions of the chromosome, including the serA region and the metE region, were mapped using pulsed field gel electrophoresis. Duplications of serA were found to be large, ranging in size from 600 kb to 2100 kb. Several of the duplications isolated at serA were caused by ectopic recombination between IS5 elements and between IS186 elements. Duplications of the metE region, however, were almost exclusively the result of ectopic recombination between ribosomal RNA cistrons. Duplication frequencies were determined at both serA and metE in wild type and mismatch repair mutant strains (mutL, mutS, uvrD and recF). Even though all of the mismatch repair mutations increased duplication frequency of metE, the largest increases were observed in the mutL and mutS strains. Duplication frequency of serA was increased less dramatically by mutations in mismatch repair. Several duplications of metE isolated in a wild type and a mismatch repair mutant were mapped. The results showed that the same repeated sequences were used for duplication formation in the mismatch repair mutant as were used in the wild type strain. Several isolates showed evidence of multiple rearrangements indicating that mismatch repair may play a role in stabilizing the genome by controlling chromosomal rearrangement. ^

A NEW TUMOR SUPPRESSOR GENE CANDIDATE REGULATED BY THE NON-CODING RNA PCA3 IN HUMAN PROSTATE CANCER

Prostate cancer is the second leading cause of cancer-related death and the most common non-skin cancer in men in the USA. Considerable advancements in the practice of medicine have allowed a significant improvement in the diagnosis and treatment of this disease and, in recent years, both incidence and mortality rates have been slightly declining. However, it is still estimated that 1 man in 6 will be diagnosed with prostate cancer during his lifetime, and 1 man in 35 will die of the disease. In order to identify novel strategies and effective therapeutic approaches in the fight against prostate cancer, it is imperative to improve our understanding of its complex biology since many aspects of prostate cancer initiation and progression still remain elusive. The study of tumor biomarkers, due to their specific altered expression in tumor versus normal tissue, is a valid tool for elucidating key aspects of cancer biology, and may provide important insights into the molecular mechanisms underlining the tumorigenesis process of prostate cancer. PCA3, is considered the most specific prostate cancer biomarker, however its biological role, until now, remained unknown. PCA3 is a long non-coding RNA (ncRNA) expressed from chromosome 9q21 and its study led us to the discovery of a novel human gene, PC-TSGC, transcribed from the opposite strand and in an antisense orientation to PCA3. With the work presented in this thesis, we demonstrate that PCA3 exerts a negative regulatory role over PC-TSGC, and we propose PC-TSGC to be a new tumor suppressor gene that contrasts the transformation of prostate cells by inhibiting Rho-GTPases signaling pathways. Our findings provide a biological role for PCA3 in prostate cancer and suggest a new mechanism of tumor suppressor gene inactivation mediated by non-coding RNA. Also, the characterization of PCA3 and PC-TSGC led us to propose a new molecular pathway involving both genes in the transformation process of the prostate, thus providing a new piece of the jigsaw puzzle representing the complex biology of prostate cancer.

MULTIPLE POSTTRANSCRIPTIONAL REGULATORY FEATURES CONTROL EXPRESSION OF ETHANOLAMINE UTILIZATION GENES IN ENTEROCOCCUS FAECALIS

Enterococcus faecalis is a Gram-positive bacterium that lives as a commensal organism in the mammalian gastrointestinal tract, but can behave as an opportunistic pathogen. Our lab discovered that mutation of the eutK gene attenuates virulence of E. faecalis in the C. elegans model host. eutK is part of the ethanolamine metabolic pathway which was previously unknown in E. faecalis. I discovered the presence of two unique posttranscriptional regulatory features that control expression of eut locus genes. The first feature I found is an AdoCBL riboswitch, a cis-acting RNA regulatory element that acts as a positive regulator of gene expression. The second feature I discovered is a unique two-component system, EutVW. The EutV response regulator contains an ANTAR family domain, which binds RNA to trigger transcriptional antitermination. I determined that induction of expression of several genes in the eut locus is dependent on ethanolamine, AdoCBL and the two-component system. AdoCBL and ethanolamine are both required for induction of eut locus gene expression. Additionally, I discovered eutG is regulated by a unique mechanism of antitermination. Both the AdoCBL riboswitch and EutV response regulator control the expression of the downstream gene eutG. EutV potentially acts through a novel antitermination mechanism in which a dimer of EutV binds to a pair of mRNA stem loops forming an antitermination complex. My data show a unique mechanism by which two environmental signals are integrated by two different posttranscriptional regulators to regulate a single locus.

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins.

Cells must rapidly sense and respond to a wide variety of potentially cytotoxic external stressors to survive in a constantly changing environment. In a search for novel genes required for stress tolerance in Saccharomyces cerevisiae, we identified the uncharacterized open reading frame YER139C as a gene required for growth at 37 degrees C in the presence of the heat shock mimetic formamide. YER139C encodes the closest yeast homolog of the human RPAP2 protein, recently identified as a novel RNA polymerase II (RNAPII)-associated factor. Multiple lines of evidence support a role for this gene family in transcription, prompting us to rename YER139C RTR1 (regulator of transcription). The core RNAPII subunits RPB5, RPB7, and RPB9 were isolated as potent high-copy-number suppressors of the rtr1Delta temperature-sensitive growth phenotype, and deletion of the nonessential subunits RPB4 and RPB9 hypersensitized cells to RTR1 overexpression. Disruption of RTR1 resulted in mycophenolic acid sensitivity and synthetic genetic interactions with a number of genes involved in multiple phases of transcription. Consistently, rtr1Delta cells are defective in inducible transcription from the GAL1 promoter. Rtr1 constitutively shuttles between the cytoplasm and nucleus, where it physically associates with an active RNAPII transcriptional complex. Taken together, our data reveal a role for members of the RTR1/RPAP2 family as regulators of core RNAPII function.

Evolution of sensory complexity recorded in a myxobacterial genome.

Myxobacteria are single-celled, but social, eubacterial predators. Upon starvation they build multicellular fruiting bodies using a developmental program that progressively changes the pattern of cell movement and the repertoire of genes expressed. Development terminates with spore differentiation and is coordinated by both diffusible and cell-bound signals. The growth and development of Myxococcus xanthus is regulated by the integration of multiple signals from outside the cells with physiological signals from within. A collection of M. xanthus cells behaves, in many respects, like a multicellular organism. For these reasons M. xanthus offers unparalleled access to a regulatory network that controls development and that organizes cell movement on surfaces. The genome of M. xanthus is large (9.14 Mb), considerably larger than the other sequenced delta-proteobacteria. We suggest that gene duplication and divergence were major contributors to genomic expansion from its progenitor. More than 1,500 duplications specific to the myxobacterial lineage were identified, representing >15% of the total genes. Genes were not duplicated at random; rather, genes for cell-cell signaling, small molecule sensing, and integrative transcription control were amplified selectively. Families of genes encoding the production of secondary metabolites are overrepresented in the genome but may have been received by horizontal gene transfer and are likely to be important for predation.

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Exon involvement in the regulation of MuSVts110 RNA splicing

MuSVts110 is a conditionally defective mutant of Moloney murine sarcoma virus which undergoes a novel tmperature-dependent splice event at growth temperatures of 33$\sp\circ$C or lower. Relative to wild-type MuSV-124, MuSVts110 contains a 1487 base deletion spanning from the 3$\sp\prime$ end of the p30 gag coding region to just downstream of the first v-mos initiation codon. As a result, the gag and mos genes are fused out of frame and no v-mos protein is expressed. However, upon a shift to 33$\sp\circ$C or lower, a splice event occurs which removes 431 bases, realigns the gag and mos genes, and allows read-through translation of a P85gag-mos transforming protein. Interestingly, while the cryptic splice sites utilized in MuSVts110 are present and unaltered in MuSV-124, they are never used. Due to the 1487 base deletion, the MuSV-124 intron was reduced from 1919 to 431 bases suggesting that intron size might be involved in the activation of these cryptic splice sites in MuSVts110. Since the splicing phenotype of the MuSVts110 equivalent (TS32 DNA) which contains the identical 1487 base deletion introduced into otherwise wild-type MuSV-124 DNA, was indistinguishable from authentic MuSVts110, it was concluded that this deletion alone is responsible for activation of the cryptic splice sites used in MuSVts110. These results also confirmed that thermodependent splicing is an intrinsic property of the viral RNA and not due to some cellular defect. Furthermore, analysis of gag gene deletion and frameshift MuSVts110 mutants demonstrated that viral gag gene proteins do not play a role in regulation of MuSVts110 splicing. Instead, cis-acting viral sequences appear to mediate regulation of the splice event.^ Our initial observation that truncation of the MuSVts110 transcript, leaving only residual amounts of the flanking exon sequences, completely abolished splicing activity argued that exon sequences might participate in the regulation of the splice event.^ Analysis of exon sequence involvement has also identified cis-acting sequences important in the thermodependence of the splice event. Data suggest that regulation of the MuSVts110 splice event involves multiple interactions between specific intron and exon sequences and spliceosome components which together limit splicing activity to temperatures of 33$\sp\circ$C or lower while simultaneously restricting splicing to a maximum of 50% efficiency. (Abstract shortened with permission of author.) ^

Mapping and cloning of genes from portions of the human genome using somatic cell hybrids

Human x rodent somatic cell hybrids have played an important role in human genetics research. They have been especially useful for assigning genes to chromosomes and isolating DNA markers from specific regions of the human genome.^ By employing a combination of somatic cell genetic, recombinant DNA, and cytogenetic techniques, human DNA excision repair gene ERCC4 was mapped regionally to human 16p13.13-13.2, even though the gene has not been cloned. Human x Chinese hamster ovary (CHO) cell hybrids selected for human ERCC4 activity and containing 16p13.1-p13.3 as the only human genetic material were identified. These hybrids were used to order DNA markers located in 16p13.1-p13.3. New DNA markers physically close to ERCC4 were isolated from such hybrids. Using amplified human DNA from the hybrids as probe in fluorescent in situ hybridization, the short arm breakpoint in the chromosome 16 inversion associated with acute myelomonocytic leukemia (AMML) was found to be physically close to the ERCC4 gene. The physical mapping and eventually, the cloning of the ERCC4 gene, will benefit the understanding of the DNA repair system and the study of other important biomedical problems such as tumorigenesis.^ To facilitate the cloning of ERCC4 gene and, in general, the cloning of genes from any defined regions of the human genome, a method was developed for the direct isolation of human transcribed genes ffom somatic cell hybrids. cDNA was prepared from human x rodent hybrid by using consensus 5$\sp\prime$ splice site sequences as primers. These primers were designed to select immature, unspliced messenger RNA (still retaining species specific repeat sequences) as templates. Screening of a derived cDNA library for human repeat sequences resulted in the isolation of human clones at the anticipated frequency with characteristics expected of exons of transcribed human genes. The usefulness of the splice site specific primers was analyzed and the cDNA synthesis conditions with these primers were optimized. The procedure was shown to be sensitive enough to clone weakly expressed genes. Studying the expression of the represented genes with the isolated clones was shown to be feasible. Such regional specific human gene fragments will be very valuable for many human genetic studies such as the search of inherited disease genes and the construction of a cDNA map of the human genome. ^

Accessing novel developmental mechanisms in the mouse by gene trapping

Genetic analysis is a powerful method for analyzing the function of specific genes in development. I sought to identify novel genes in the mouse using a genetic analysis relying on the expression pattern and phenotype of mutated genes. To this end, I have conducted a gene trap screen using the vector $\rm SA\beta geo,$ a promoterless DNA construct that encodes a fusion protein with lacZ and neomycin resistance activities. Productive integration and expression of the $\beta$geo protein in embryonic stem (ES) cells requires integration into an active transcription unit. The endogenous regulatory elements direct reporter gene expression which reflects the expression of the endogenous gene. Of eight mouse lines generated from gene trap ES cell clones, four showed differential regulation of $\beta$geo activity during embryogenesis. These four were analyzed in more detail.^ Three of the lines RNA 1, RNA2 and RNA 3 had similar expression patterns, within subsets of cells in sites of embryonic hematopoiesis. Cloning of the trapped genes revealed that all three integrations had occurred within 45S rRNA precursor transcription units. These results imply that there exists in these cells some mechanism responsible for the efficient production of the $\beta$geo protein from an RNA polymerase I transcript that is not present in most of the cells in the embryo.^ The fourth line, GT-2, showed widespread, dynamic expression. Many of the sites of expression were important classic embryonic induction systems. Cloning of the sequences fused to the $5\sp\prime$ end of the $\beta$geo sequence revealed that the trapped gene contained significant sequence homology with a previously identified human sequence HumORF5. An open reading frame of this sequence is homologous to a group of eukaryotic proteins that are members of the RNA helicase superfamily I.^ Analysis of the gene trap lines suggests that potentially novel developmental mechanisms have been uncovered. In the case of RNA 1, 2 and 3, the differential production of ribosomal RNAs may be required for differentiation or function of the $\beta$geo positive hematopoietic cells. In the GT-2 line, a previously unsuspected temporal and spatial regulation of a putative RNA helicase implies a role for this activity during specific aspects of mouse development. ^