Disruption of the bacterial OLE RNP complex impairs growth on alternative carbon sources.

Ornate, large, extremophilic (OLE) RNAs comprise a class of large noncoding RNAs in bacteria whose members form a membrane-associated ribonucleoprotein (RNP) complex. This complex facilitates cellular adaptation to diverse stresses such as exposure to cold, short-chain alcohols, and elevated Mg2+ concentrations. Here, we report additional phenotypes exhibited by Halalkalibacterium halodurans (formerly called Bacillus halodurans) strains lacking functional OLE RNP complexes. Genetic disruption of the complex causes restricted growth compared to wild-type cells when cultured in minimal media (MM) wherein glucose is replaced with alternative carbon/energy sources. Genetic suppressor selections conducted in glutamate MM yielded isolates that carry mutations in or near genes relevant to Mn2+ homeostasis (ykoY and mntB), phosphate homeostasis (phoR), and putative multidrug resistance (bmrCD). These functional links between OLE RNA, carbon/energy management, and other fundamental processes including protein secretion are consistent with the hypothesis that the OLE RNP complex is a major contributor to cellular adaptation to unfavorable growth conditions.

Evidence that OLE RNA is a component of a major stress-responsive ribonucleoprotein particle in extremophilic bacteria.

OLE RNA is a ~600-nucleotide noncoding RNA present in many Gram-positive bacteria that thrive mostly in extreme environments, including elevated temperature, salt, and pH conditions. The precise biochemical functions of this highly conserved RNA remain unknown, but it forms a ribonucleoprotein (RNP) complex that localizes to cell membranes. Genetic disruption of the RNA or its essential protein partners causes reduced cell growth under various stress conditions. These phenotypes include sensitivity to short-chain alcohols, cold intolerance, reduced growth on sub-optimal carbon sources, and intolerance of even modest concentrations of Mg2+ . Thus, many bacterial species appear to employ OLE RNA as a component of an intricate RNP apparatus to monitor fundamental cellular processes and make physiological and metabolic adaptations. Herein we hypothesize that the OLE RNP complex is functionally equivalent to the eukaryotic TOR complexes, which integrate signals from various diverse pathways to coordinate processes central to cell growth, replication, and survival.

Ornate, large, extremophilic (OLE) RNA forms a kink turn necessary for OapC protein recognition and RNA function.

Ornate, large, extremophilic (OLE) RNAs represent a class of noncoding RNAs prevalent in Gram-positive, extremophilic/anaerobic bacterial species. OLE RNAs (∼600 nt), whose precise biochemical functions remain mysterious, form an intricate secondary structure interspersed with regions of highly conserved nucleotides. In the alkali-halophilic bacterium Bacillus halodurans, OLE RNA is a component of a ribonucleoprotein (RNP) complex involving at least two proteins named OapA and OapB, but additional components may exist that could point to functional roles for the RNA. Disruption of the genes for either OLE RNA, OapA, or OapB result in the inability of cells to overcome cold, alcohol, or Mg2+ stresses. In the current study, we used in vivo crosslinking followed by OLE RNA isolation to identify the protein YbxF as a potential additional partner in the OLE RNP complex. Notably, a mutation in the gene for this same protein was also reported to be present in a strain wherein the complex is nonfunctional. The B. halodurans YbxF (herein renamed OapC) is homologous to a bacterial protein earlier demonstrated to bind kink turn (k-turn) RNA structural motifs. In vitro RNA-protein binding assays reveal that OLE RNA forms a previously unrecognized k-turn that serves as the natural binding site for YbxF/OapC. Moreover, B. halodurans cells carrying OLE RNAs with disruptive mutations in the k-turn exhibit phenotypes identical to cells lacking functional OLE RNP complexes. These findings reveal that the YbxF/OapC protein of B. halodurans is important for the formation of a functional OLE RNP complex.

Architectures and complex functions of tandem riboswitches.

Riboswitch architectures that involve the binding of a single ligand to a single RNA aptamer domain result in ordinary dose-response curves that require approximately a 100-fold change in ligand concentration to cover nearly the full dynamic range for gene regulation. However, by using multiple riboswitches or aptamer domains in tandem, these ligand-sensing structures can produce additional, complex gene control outcomes. In the current study, we have computationally searched for tandem riboswitch architectures in bacteria to provide a more complete understanding of the diverse biological and biochemical functions of gene control elements that are made exclusively of RNA. Numerous different arrangements of tandem homologous riboswitch architectures are exploited by bacteria to create more 'digital' gene control devices, which operate over a narrower ligand concentration range. Also, two heterologous riboswitch aptamers are sometimes employed to create two-input Boolean logic gates with various types of genetic outputs. These findings illustrate the sophisticated genetic decisions that can be made by using molecular sensors and switches based only on RNA.

Primary Care Practitioner Perceptions on the Follow-up of Abnormal Cancer Screening Test Results.

Importance: Health care systems focus on delivering routine cancer screening to eligible individuals, yet little is known about the perceptions of primary care practitioners (PCPs) about barriers to timely follow-up of abnormal results. Objective: To describe PCP perceptions about factors associated with the follow-up of abnormal breast, cervical, colorectal, and lung cancer screening test results. Design, Setting, and Participants: Survey study of PCPs from 3 primary care practice networks in New England between February and October 2020, prior to participating in a randomized clinical trial to improve follow-up of abnormal cancer screening test results. Participants were physicians and advanced practice clinicians from participating practices. Main Outcomes and Measures: Self-reported process, attitudes, knowledge, and satisfaction about the follow-up of abnormal cancer screening test results. Results: Overall, 275 (56.7%) PCPs completed the survey (range by site, 34.9%-71.9%) with more female PCPs (61.8% [170 of 275]) and general internists (73.1% [201 of 275]); overall, 28,7% (79 of 275) were aged 40 to 49 years. Most PCPs felt responsible for managing abnormal cancer screening test results with the specific cancer type being the best factor (range, 63.6% [175 of 275] for breast to 81.1% [223 of 275] for lung; P < .001). The PCPs reported limited support for following up on overdue abnormal cancer screening test results. Standard processes such as automated reports, reminder letters, or outreach workers were infrequently reported. Major barriers to follow-up of abnormal cancer screening test results across all cancer types included limited electronic health record tools (range, 28.5% [75 of 263]-36.5%[96 of 263]), whereas 50% of PCPs felt that there were major social barriers to receiving care for abnormal cancer screening test results for colorectal cancer. Fewer than half reported being very satisfied with the process of managing abnormal cancer screening test results, with satisfaction being greatest for breast cancer (46.9% [127 of 271]) and lowest for cervical (21.8% [59 of 271]) and lung cancer (22.4% [60 of 268]). Conclusions and Relevance: In this survey study of PCPs, important deficiencies in systems for managing abnormal cancer screening test results were reported. These findings suggest a need for comprehensive organ-agnostic systems to promote timely follow-up of abnormal cancer screening results using a primary care-focused approach across the range of cancer screening tests.

Natural Language Processing to Identify Abnormal Breast, Lung, and Cervical Cancer Screening Test Results from Unstructured Reports to Support Timely Follow-up.

Cancer screening and timely follow-up of abnormal results can reduce mortality. One barrier to follow-up is the failure to identify abnormal results. While EHRs have coded results for certain tests, cancer screening results are often stored in free-text reports, which limit capabilities for automated decision support. As part of the multilevel Follow-up of Cancer Screening (mFOCUS) trial, we developed and implemented a natural language processing (NLP) tool to assist with real-time detection of abnormal cancer screening test results (including mammograms, low-dose chest CT scans, and Pap smears) and identification of gynecological follow-up for higher risk abnormalities (i.e. colposcopy) from free-text reports. We demonstrate the integration and implementation of NLP, within the mFOCUS system, to improve the follow-up of abnormal cancer screening results in a large integrated healthcare system. The NLP pipelines have detected scenarios when guideline-recommended care was not delivered, in part because the provider mis-identified the text-based result reports.

Multilevel Follow-up of Cancer Screening (mFOCUS): Protocol for a multilevel intervention to improve the follow-up of abnormal cancer screening test results.

INTRODUCTION: While substantial attention is focused on the delivery of routine preventive cancer screening, less attention has been paid to systematically ensuring that there is timely follow-up of abnormal screening test results. Barriers to completion of timely follow-up occur at the patient, provider, care team and system levels. METHODS: In this pragmatic cluster randomized controlled trial, primary care sites in three networks are randomized to one of four arms: (1) standard care, (2) "visit-based" reminders that appear in a patient's electronic health record (EHR) when it is accessed by either patient or providers (3) visit based reminders with population health outreach, and (4) visit based reminders, population health outreach, and patient navigation with systematic screening and referral to address social barriers to care. Eligible patients in participating practices are those overdue for follow-up of an abnormal results on breast, cervical, colorectal and lung cancer screening tests. RESULTS: The primary outcome is whether an individual receives follow-up, specific to the organ type and screening abnormality, within 120 days of becoming eligible for the trial. Secondary outcomes assess the effect of intervention components on the patient and provider experience of obtaining follow-up care and the delivery of the intervention components. CONCLUSIONS: This trial will provide evidence for the role of a multilevel intervention on improving the follow-up of abnormal cancer screening test results. We will also specifically assess the relative impact of the components of the intervention, compared to standard care. TRIAL REGISTRATION: ClinicalTrials.gov NCT03979495.

Patient, provider, and clinic factors associated with the use of cervical cancer screening.

Cervical cancer screening delivery remains suboptimal. Understanding the multiple influences on use of screening is important to designing interventions. We describe the influence of patient, primary care provider (PCP), and clinic characteristics on whether a woman is up-to-date with cervical screening as of December 2016. PCPs (n = 194) and their female screen-eligible patients age 21-65 years (n = 32,115) were included in this cross-sectional analysis of patients from two primary care networks linked to a contemporaneous PCP survey. Principal independent variables for patients included: age, race, insurance, continuity of care; for PCP included: overall satisfaction with the practice of medicine, gender, hours worked per week, financial support for achieving clinical targets; and for clinic included: routine receipt of data on preventive care performance and language translation resources. Overall, 66.6% of women were up-to-date. Women were less likely to be up-to-date with cervical cancer screening if they were younger and were more likely to be screened if they were Black, Hispanic or Asian vs. White. Women with greater continuity of primary care or with a female PCP were more likely to be up-to-date (1.52; 1.33-1.75); those who received care in a clinic that was less prepared to manage language translation were less likely to be up-to-date (0.78; 0.65-0.95). Patient, provider, and clinic factors all influence use of cervical cancer screening. Systems interventions like improving continuity of care, promoting translation services, or enhanced efforts to track screening among patients of male PCPs may improve delivery.

Structure of a bacterial OapB protein with its OLE RNA target gives insights into the architecture of the OLE ribonucleoprotein complex.

The OLE (ornate, large, and extremophilic) RNA class is one of the most complex and well-conserved bacterial noncoding RNAs known to exist. This RNA is known to be important for bacterial responses to stress caused by short-chain alcohols, cold, and elevated Mg2+ concentrations. These biological functions have been shown to require the formation of a ribonucleoprotein (RNP) complex including at least two protein partners: OLE-associated protein A (OapA) and OLE-associated protein B (OapB). OapB directly binds OLE RNA with high-affinity and specificity and is believed to assist in assembling the functional OLE RNP complex. To provide the atomic details of OapB-OLE RNA interaction and to potentially reveal previously uncharacterized protein-RNA interfaces, we determined the structure of OapB from Bacillus halodurans alone and in complex with an OLE RNA fragment at resolutions of 1.0 Å and 2.0 Å, respectively. The structure of OapB exhibits a K-shaped overall architecture wherein its conserved KOW motif and additional unique structural elements of OapB form a bipartite RNA-binding surface that docks to the P13 hairpin and P12.2 helix of OLE RNA. These high-resolution structures elucidate the molecular contacts used by OapB to form a stable RNP complex and explain the high conservation of sequences and structural features at the OapB-OLE RNA-binding interface. These findings provide insight into the role of OapB in the assembly and biological function of OLE RNP complex and can guide the exploration of additional possible OLE RNA-binding interactions present in OapB.

Bacillus halodurans OapB forms a high-affinity complex with the P13 region of the noncoding RNA OLE.

Noncoding RNAs (ncRNAs) longer than 200 nucleotides are rare in bacteria, likely because bacterial genomes are under strong evolutionary pressures to maintain a small genome size. Of the long ncRNAs unique to bacteria, the OLE (ornate, large, extremophilic) RNA class is among the largest and most structurally complex. OLE RNAs form a ribonucleoprotein (RNP) complex by partnering with at least two proteins, OapA and OapB, that directly bind OLE RNA. The biochemical functions of the OLE RNP complex remain unknown, but are required for proper adaptation to certain environmental stresses, such as cold temperatures, short chain alcohols, and high magnesium concentrations. In the current study, we used electrophoretic mobility shift assays to examine the binding of OLE RNA fragments by OapB and found that OapB recognizes a small subregion of OLE RNA, including stem P13, with a dissociation constant (KD ) of ∼700 pm Analyses with mutated RNA constructs, and the application of in vitro selection, revealed that strong binding of OLE RNA by OapB requires a stem containing a precisely located single-nucleotide bulge and a GNRA tetraloop. Although the vast majority of bacteria with the ole gene also have the oapB gene, there are many whose genomes contain oapB but lack ole, suggesting that OapB has other RNA partners in some species that might exhibit similar structural features.

Disruption of the OLE ribonucleoprotein complex causes magnesium toxicity in Bacillus halodurans.

OLE RNAs represent an unusual class of bacterial noncoding RNAs common in Gram-positive anaerobes. The OLE RNA of the alkaliphile Bacillus halodurans is highly expressed and naturally interacts with at least two RNA-binding proteins called OapA and OapB. The phenotypes of the corresponding knockouts include growth inhibition when exposed to ethanol or other short-chain alcohols or when incubated at modestly reduced temperatures (e.g. 20°C). Intriguingly, the OapA 'PM1' mutant, which carries two amino acid changes to a highly conserved region, yields a dominant-negative phenotype that causes more severe growth defects under these same stress conditions. Herein, we report that the PM1 strain also exhibits extreme sensitivity to elevated Mg2+ concentrations, beginning as low as 2 mM. Suppressor mutants predominantly map to genes for aconitate hydratase and isocitrate dehydrogenase, which are expected to alter cellular citrate concentrations. Citrate reduces the severity of the Mg2+ toxicity phenotype, but neither the genomic mutations nor the addition of citrate to the medium overcomes ethanol toxicity or temperature sensitivity. These findings reveal that OLE RNA and its protein partners are involved in biochemical responses under several stress conditions, wherein the unusual sensitivity to Mg2+ can be independently suppressed by specific genomic mutations.

Randomized Trial of Personalized Breast Density and Breast Cancer Risk Notification.

BACKGROUND: Despite widespread implementation of mammographic breast density (MBD) notification laws, the impact of these laws on knowledge of MBD and knowledge of breast cancer risk is limited by the lack of tools to promote informed decision-making in practice. OBJECTIVE: To develop and evaluate whether brief, personalized informational videos following a normal mammogram in addition to a legislatively required letter about MBD result can improve knowledge of MBD and breast cancer risk compared to standard care (i.e., legislatively required letter about MBD included with the mammogram result). DESIGN/PARTICIPANTS: Prospective randomized controlled trial of English-speaking women, age 40-74 years, without prior history of breast cancer, receiving a screening mammogram with a normal or benign finding (intervention group n = 235, control group n = 224). INTERVENTION: brief (3-5 min) video, personalized to a woman's MBD result and breast cancer risk. MAIN MEASURES: Primary outcomes were a woman's knowledge of her MBD and risk of breast cancer. Secondary outcomes included whether a woman reported that she discussed the results of her mammogram with her primary care provider (PCP). KEY RESULTS: Relative to women in the control arm, women in the intervention arm had greater improvement in their knowledge of both their personal MBD (intervention pre/post 39.2%/ 77.5%; control pre/post 36.2%/ 37.5%; odds ratio (OR) 5.34 for change for intervention vs. control, 95% confidence interval (CI) 3.87-7.36; p < 0.001) and risk of breast cancer (intervention pre/post: 66.8%/74.0%; control pre/post 67.9%/ 65.2%; OR 1.42, 95% confidence interval (CI) 1.09-1.84; p = 0.01). Women in the intervention group were more likely than those in the control group to report discussing the results of their mammogram with their PCP (p = 0.05). CONCLUSIONS: Brief, personalized videos following mammography can improve knowledge of MBD and personal risk of breast cancer compared to a legislatively mandated informational letter. Trial Registration Clinicaltrials.gov (NCT02986360).

Large Noncoding RNAs in Bacteria.

Bacterial noncoding RNA (ncRNA) classes longer than 200 nucleotides are rare but are responsible for performing some of the most fundamental tasks in living cells. RNAs such as 16S and 23S rRNA, group I and group II introns, RNase P ribozymes, transfer-messenger RNAs, and coenzyme B12 riboswitches are diverse in structure and accomplish biochemical functions that rival the activities of proteins. Over the last decade, a number of new classes of large ncRNAs have been uncovered in bacteria. A total of 21 classes with no established functions have been identified through the use of bioinformatics search strategies. Based on precedents for bacterial large ncRNAs performing sophisticated functions, it seems likely that some of these structured ncRNAs also will prove to carry out complex functions. Thus, determining their roles will provide a better understanding of fundamental biological processes. A few studies have produced data that provide clues to the purposes of some of these recently found classes, but the true functions of most classes remain mysterious.

A second RNA-binding protein is essential for ethanol tolerance provided by the bacterial OLE ribonucleoprotein complex.

OLE (ornate, large, extremophilic) RNAs comprise a class of structured noncoding RNAs (ncRNAs) found in many extremophilic bacteria species. OLE RNAs constitute one of the longest and most widespread bacterial ncRNA classes whose major biochemical function remains unknown. In the Gram-positive alkaliphile Bacillus halodurans, OLE RNA is abundant, and localizes to the cell membrane by association with the transmembrane OLE-associated protein called OapA (formerly OAP). These characteristics, along with the well-conserved sequence and structural features of OLE RNAs, suggest that the OLE ribonucleoprotein (RNP) complex performs important biological functions. B. halodurans strains lacking OLE RNA (∆ole) or OapA (∆oapA) are less tolerant of cold (20 °C) and short-chain alcohols (e.g., ethanol). Here, we describe the effects of a mutant OapA (called PM1) that more strongly inhibits growth under cold or ethanol stress compared with strains lacking the oapA gene, even when wild-type OapA is present. This dominant-negative effect of PM1 is reversed by mutations that render OLE RNA nonfunctional. This finding demonstrates that the deleterious PM1 phenotype requires an intact RNP complex, and suggests that the complex has one or more additional undiscovered components. A genetic screen uncovered PM1 phenotype suppressor mutations in the ybzG gene, which codes for a putative RNA-binding protein of unknown biological function. We observe that YbzG protein (also called OapB) selectively binds OLE RNA in vitro, whereas a mutant version of the protein is not observed to bind OLE RNA. Thus, YbzG/OapB is an important component of the functional OLE RNP complex in B. halodurans.

Challenges of ligand identification for the second wave of orphan riboswitch candidates.

Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates. This facilitated the validation of riboswitches for the signaling molecules c-di-AMP, ZTP, and ppGpp, the metal ion Mn2+, and the metabolites guanidine and PRPP. Such studies also yield useful linkages between the ligands sensed by the riboswitches and numerous biochemical pathways. In the current report, we describe the known characteristics of 30 distinct classes of orphan riboswitch candidates - some of which have remained unsolved for over a decade. We also discuss the prospects for uncovering novel biological insights via focused studies on these RNAs. Lastly, we make recommendations for experimental objectives along the path to finding ligands for these mysterious RNAs.

Diffusion of digital breast tomosynthesis among women in primary care: associations with insurance type.

Digital breast tomosynthesis (DBT) has shown potential to improve breast cancer screening and diagnosis compared to digital mammography (DM). The FDA approved DBT use in conjunction with conventional DM in 2011, but coverage was approved by CMS recently in 2015. Given changes in coverage policies, it is important to monitor diffusion of DBT by insurance type. This study examined DBT trends and estimated associations with insurance type. From June 2011 to September 2014, DBT use in 22 primary care centers in the Dartmouth -Brigham and Women's Hospital Population-based Research Optimizing Screening through Personalized Regimens research center (PROSPR) was examined among women aged 40-89. A longitudinal repeated measures analysis estimated the proportion of DBT performed for screening or diagnostic indications over time and by insurance type. During the study period, 93,182 mammograms were performed on 48,234 women. Of these exams, 16,506 DBT tests were performed for screening (18.1%) and 2537 were performed for diagnosis (15.7%). Between 2011 and 2014, DBT utilization increased in all insurance groups. However, by the latest observed period, screening DBT was used more frequently under private insurance (43.4%) than Medicaid (36.2%), Medicare (37.8%), other (38.6%), or no insurance (32.9%; P < 0.0001). No sustained differences in use of DBT for diagnostic testing were seen by insurance type. DBT is increasingly used for breast cancer screening and diagnosis. Use of screening DBT may be associated with insurance type. Surveillance is required to ensure that disparities in breast cancer screening are minimized as DBT becomes more widely available.

Challenges With Identifying Indication for Examination in Breast Imaging as a Key Clinical Attribute in Practice, Research, and Policy.

PURPOSE: To assess indication for examination for four breast imaging modalities and describe the complexity and heterogeneity of data sources and ascertainment methods. METHODS: Indication was evaluated among the Population-based Research Optimizing Screening through Personalized Regimens (PROSPR) breast cancer research centers (PRCs). Indication data were reported overall and separately for four breast imaging modalities: digital mammography (DM), digital breast tomosynthesis (DBT), ultrasound (US), and magnetic resonance imaging (MRI). RESULTS: The breast PRCs contributed 236,262 women with 607,735 breast imaging records from 31 radiology facilities. We found a high degree of heterogeneity for indication within and across six data sources. Structured codes within a data source were used most often to identify indication for mammography (59% DM, 85% DBT) and text analytics for US (45%) and MRI (44%). Indication could not be identified for 17% of US and 26% of MRI compared with 2% of mammography examinations (1% DM, 3% DBT). CONCLUSIONS: Multiple and diverse data sources, heterogeneity of ascertainment methods, and nonstandardization of codes within and across data systems for determining indication were found. Consideration of data sources and standardized methodology for determining indication is needed to assure accurate measurement of cancer screening rates and performance in clinical practice and research.

Patients with Refractory Crohn's Disease Successfully Treated with Ustekinumab.

BACKGROUND: Ustekinumab is a new biologic therapy targeting interleukin-12 and interleukin -23. It is currently approved for the treatment of psoriasis, but clinical trials have shown that it can induce and maintain remission in Crohn's disease (CD). We aim to evaluate effectiveness of ustekinumab in the treatment of CD. METHODS: A retrospective chart review was performed including patients (pts) from 2 academic medical centers with complicated, refractory CD started on ustekinumab between June 2011 and June 2014. Pts were treated based on a novel subcutaneous dosing schedule designed to simulate the intravenous load used in clinical trials. RESULTS: Forty-five pts were treated with ustekinumab during this study period. Of the pts who had clinical parameters available before and after medication start, 46% achieved clinical response (Harvey-Bradshaw index decrease ≥ 3) and 35% achieved clinical remission (Harvey-Bradshaw index ≤ 3). Short inflammatory bowel disease questionnaire scores increased significantly (46 [20, 68] to 55 [32, 70], P < 0.05). Erythrocyte sedimentation rate decreased significantly (20 [3, 54] to 12 [0, 42] mm/h, P < 0.05). C-reactive protein decreased significantly (4.9 [0.3, 111] to 3.3 [0.2, 226] mg/L, P < 0.05). Seventy-six percent of patients demonstrated an endoscopic response and 24% achieved complete endoscopic remission. Twelve patients (26%) were hospitalized for IBD-related issues. Four pts had infection-related complications. Six pts (13%) underwent surgery for IBD-related issues. Three pts stopped ustekinumab, 1 for pt preference and 2 for the lack of response. CONCLUSIONS: Using a novel subcutaneous dosing schedule, ustekinumab was successful in improving clinical, laboratory, and endoscopic markers of disease activity in patients with severe, refractory CD.

Biochemical analysis of pistol self-cleaving ribozymes.

Pistol RNAs are members of a distinct class of self-cleaving ribozymes that was recently discovered by using a bioinformatics search strategy. Several hundred pistol ribozymes share a consensus sequence including 10 highly conserved nucleotides and many other modestly conserved nucleotides associated with specific secondary structure features, including three base-paired stems and a pseudoknot. A representative pistol ribozyme from the bacterium Lysinibacillus sphaericus was found to promote RNA strand scission with a rate constant of ∼10 min(-1) under physiological Mg(2+) and pH conditions. The reaction proceeds via the nucleophilic attack of a 2'-oxygen atom on the adjacent phosphorus center, and thus adheres to the same general catalytic mechanism of internal phosphoester transfer as found with all other classes of natural self-cleaving ribozymes discovered to date. Analyses of the kinetic characteristics and the metal ion requirements of the cleavage reaction reveal that members of this ribozyme class likely use several catalytic strategies to promote the rapid cleavage of RNA.

Biochemical analysis of hatchet self-cleaving ribozymes.

Hatchet RNAs are members of a novel self-cleaving ribozyme class that was recently discovered by using a bioinformatics search strategy. The consensus sequence and secondary structure of this class includes 13 highly conserved and numerous other modestly conserved nucleotides interspersed among bulges linking four base-paired substructures. A representative hatchet ribozyme from a metagenomic source requires divalent ions such as Mg(2+) to promote RNA strand scission with a maximum rate constant of ∼4 min(-1). As with all other small self-cleaving ribozymes discovered to date, hatchet ribozymes employ a general mechanism for catalysis involving the nucleophilic attack of a ribose 2'-oxygen atom on an adjacent phosphorus center. Kinetic characteristics of the reaction demonstrate that members of this ribozyme class have an essential requirement for divalent metal ions and that they might have a complex active site that employs multiple catalytic strategies to accelerate RNA cleavage by internal phosphoester transfer.