Classification of the nucleolytic ribozymes based upon catalytic mechanism

The nucleolytic ribozymes carry out site-specific RNA cleavage reactions by nucleophilic attack of the 2’-oxygen atom on the adjacent phosphorus with an acceleration of a million-fold or greater. A major part of this arises from concerted general acid-base catalysis. Recent identification of new ribozymes has expanded the group to a total of nine and this provides a new opportunity to identify sub-groupings according to the nature of the general base and acid. These include nucleobases, hydrated metal ions, and 2’-hydroxyl groups. Evolution has selected a number of different combinations of these elements that lead to efficient catalysis. These differences provide a new mechanistic basis for classifying these ribozymes

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field

Ribozyme-based insulator parts buffer synthetic circuits from genetic context

Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as 'insulator parts', is screened in Escherichia coli. We find that ribozymes that cleave the 5′ untranslated region (5′-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs.Life Technologies, Inc.United States. Defense Advanced Research Projects Agency (DARPA CLIO N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)National Science Foundation (U.S.) (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SynBERC, SA5284-11210

Does clinical examination aid in the diagnosis of urinary tract infections in women? A systematic review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Clinicians should be aware of the diagnostic values of various symptoms, signs and antecedents. This information is particularly important in primary care settings, where sophisticated diagnostic approaches are not always feasible. The aim of the study is to determine the probability that various symptoms, signs, antecedents and tests predict urinary tract infection (UTI) in women.</p> <p>Methods</p> <p>We conducted a systematic search of the MEDLINE and EMBASE databases to identify articles published in all languages through until December 2008. We particularly focused on studies that examined the diagnostic accuracy of at least one symptom, sign or patient antecedent related to the urinary tract. We included studies where urine culture, a gold standard, was preformed by primary care providers on female subjects aged at least 14 years. A meta-analysis of the likelihood ratio was performed to assess variables related to the urinary tract symptoms.</p> <p>Results</p> <p>Of the 1, 212 articles identified, 11 met the selection criteria. Dysuria, urgency, nocturia, sexual activity and urgency with dysuria were weak predictors of urinary tract infection, whereas increases in vaginal discharge and suprapubic pain were weak predictors of the absence of infection. Nitrites or leukocytes in the dipstick test are the only findings that clearly favored a diagnosis of UTI.</p> <p>Conclusions</p> <p>Clinical findings do not aid in the diagnosis of UTI among women who present with urinary symptoms. Vaginal discharge is a weak indicator of the absence of infection. The urine dipstick test was the most reliable tool for detecting UTI.</p

Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA.

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication

Autolytic processing of a phosphorothioate diester bond.

A small satellite RNA of tobacco ringspot virus replicates in tissues infected with tobacco ringspot virus and accumulates in virus capsids, forming virus-like particles. Previous research showed that multimeric forms of this satellite RNA have tandem repeats of the "monomeric" satellite RNA sequence of 359 or 360 nucleotide residues. The multimeric RNAs undergo autolytic processing at a specific CpA phosphodiester bond, the junction, to generate the monomeric RNA. We substituted phosphorothioate diester bonds for various sets of phosphodiester bonds, in dimeric and truncated forms of the satellite RNA. The degree of reduction in autolytic cleavage varied both with the sites of substitution and the size of the RNA molecules. Analyses of a product of the autolysis reaction suggest that one phosphorothioate diester bond most strongly interferes with processing, the one introduced at the CpA junction during its synthesis from adenosine-5'-0-(1-thiotriphosphate). However, extensive introduction of phosphorothioate diester bonds elsewhere in the molecule also decreased processing, possibly by altering conformation

Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration.

Both polarities of the satellite RNA of tobacco ringspot virus occur in infected cells in multimeric forms which are capable of autolytic processing, using different sequences and structures [Feldstein, P.A., et al., Proc. Nat. Acad. Sci. USA (1990) 87 (in press)]. These transesterification reactions generate a 2',3'-cyclophosphate and a 5'-hydroxyl as the two new end groups. Cleavage is at a CpA for the (+) polarity RNA and at an ApG for the (-) polarity RNA. We enzymically synthesized oligoribonucleotides with processing capability and with specific 35S-labeled phosphorothioate diesters in the Rp configuration. After processing had occurred, the terminal nucleoside-2',3'-cyclophosphorothioate diester residues were recovered from the appropriate product by digestion with nuclease and phosphatase. Comparisons with specially prepared endo- and exoisomer reference compounds by thin layer chromatography and autoradiography revealed that the [35S]cytidine- and [35S]adenosine-2',3'-cyclophosphorothioate both were endo-isomers. The results are consistent with transesterification occurring by an inline SN2(P) attack of the 2'-hydroxyl group in the autolytic processing reactions of both polarities of the satellite RNA