Colin A. Smith

Induced folding in RNA-protein recognition: more than a simple molecular handshake.

Finally, we consider a few possible advantages of using unfolded or partially folded proteins to recognize RNA, though they are by no means restricted to RNA–protein interactions. As described for N, maintaining proteins in an unfolded state provides the opportunity to monitor the bound (or functional) status of a protein, using proteases to regulate protein concentration. Recognition of a disordered protein may allow interactions with multiple partners, each appropriately molding a binding surface to its own needs, as described for nuclear receptor dimerization and as is thought to occur with disordered activation domains of transcription factors. Induced folding or the ability to remodel surfaces can facilitate the ordered addition of components or allow signaling of binding events to other partners; for example, both the N–boxB and Ffh–4.5S RNA complexes provide new recognition surfaces for other proteins or domains. This feature is expected to be especially important for large ribonucleoprotein complexes such as the ribosome or spliceosome, in which many components must be accurately assembled or ordered events must take place. The ability to remodel also might facilitate ordered exchanges with multiple partners, as might occur in pre-mRNA splicing, and the disordered state itself might provide important nonspecific RNA contacts at appropriate times. Some interactions are topologically impossible using two rigid surfaces, and full specificity may be achieved only through induced fit mechanisms. For example, an RNA site cannot be surrounded using a preformed protein structure; in DNA–protein complexes, encircling often is achieved using flexible protein arms. From an evolutionary perspective, many sequences are expected to form disordered domains and some will contain adaptable interaction surfaces. During the transition from an RNA world to a protein-based world, flexible or even unfolded structures might well have provided adequate affinities and specificities to evolve the necessary functions.

An RNA-binding chameleon.

The arginine-rich RNA binding motif is found in a wide variety of proteins, including several viral regulatory proteins. Although related at the primary sequence level, arginine-rich domains from different proteins adopt different conformations depending on the RNA site recognized, and in some cases fold only in the context of RNA. Here we show that the RNA binding domain of the Jembrana disease virus (JDV) Tat protein is able to recognize two different TAR RNA sites, from human and bovine immunodeficiency viruses (HIV and BIV, respectively), adopting different conformations in the two RNA contexts and using different amino acids for recognition. In addition to the conformational differences, the JDV domain requires the cyclin T1 protein for high-affinity binding to HIV TAR, but not to BIV TAR. The chameleon-like behavior of the JDV Tat RNA binding domain reinforces the concept that RNA molecules can provide structural scaffolds for protein folding, and suggests mechanisms for evolving distinct RNA binding specificities from a single multifunctional domain.

The ability of a variety of polymerases to synthesize past site-specific cis-syn, trans-syn-II, (6-4), and Dewar photoproducts of thymidylyl- (3→5')- thymidine

The role of photoproduct structure, 3′ → 5′ exonuclease activity, and processivity on polynucleotide synthesis past photoproducts of thymidylyl-(3′ → 5′)-thymidine was investigated. Both Moloney murine leukemia virus reverse transcriptase and 3′ → 5′ exonuclease-deficient (exo−) Vent polymerase were blocked by all photoproducts, whereas Taq polymerase could slowly bypass the cis-syn dimer. T7 RNA polymerase was able to bypass all the photoproducts in the order cis-syn> Dewar > (6-4) > trans-syn-II. Klenow fragment could not bypass any of the photoproducts, but an exo−mutant could bypass the cis-syn dimer to a greater extent than the others. Likewise T7 DNA polymerase, composed of the T7 gene 5 protein and Escherichia coli thioredoxin, was blocked by all the photoproducts, but the exo− mutant Sequenase 2.0 was able to bypass them all in the order cis-syn > Dewar > trans-syn-II > (6-4). No bypass occurred with an exo− gene 5 protein in the absence of the thioredoxin processivity factor. Bypass of the cis-syn andtrans-syn-II products by Sequenase 2.0 was essentially non-mutagenic, whereas about 20% dTMP was inserted opposite the 5′-T of the Dewar photoproduct. A mechanism involving a transient abasic site is proposed to account for the preferential incorporation of dAMP opposite the 3′-T of the photoproducts.

A Web resource for standardized benchmark datasets, metrics, and rosetta protocols for macromolecular modeling and design

The development and validation of computational macromolecular modeling and design methods depend on suitable benchmark datasets and informative metrics for comparing protocols. In addition, if a method is intended to be adopted broadly in diverse biological applications, there needs to be information on appropriate parameters for each protocol, as well as metrics describing the expected accuracy compared to experimental data. In certain disciplines, there exist established benchmarks and public resources where experts in a particular methodology are encouraged to supply their most efficient implementation of each particular benchmark. We aim to provide such a resource for protocols in macromolecular modeling and design. We present a freely accessible web resource (https://kortemmelab.ucsf.edu/benchmarks) to guide the development of protocols for protein modeling and design. The site provides benchmark datasets and metrics to compare the performance of a variety of modeling protocols using different computational sampling methods and energy functions, providing a “best practice” set of parameters for each method. Each benchmark has an associated downloadable benchmark capture archive containing the input files, analysis scripts, and tutorials for running the benchmark. The captures may be run with any suitable modeling method; we supply command lines for running the benchmarks using the Rosetta software suite. We have compiled initial benchmarks for the resource spanning three key areas: prediction of energetic effects of mutations, protein design, and protein structure prediction, each with associated state-of-the-art modeling protocols. With the help of the wider macromolecular modeling community, we hope to expand the variety of benchmarks included on the website and continue to evaluate new iterations of current methods as they become available.

Microbial transformation of nandrolone with Cunninghamella echinulata and Cunninghamella blakesleeana and evaluation of leishmaniacidal activity of transformed products

Therapeutic potential of nandrolone and its derivatives against leishmaniasis has been studied. A number of derivatives of nandrolone (1) were synthesized through biotransformation. Microbial transformation of nandrolone (1) with Cunninghamella echinulata and Cunninghamella blakesleeana yielded three new metabolites, 10β,12β,17β-trihydroxy-19-nor-4-androsten-3-one (2), 10β,16α,17β-trihydroxy-19-nor-4-androsten-3-one (3), and 6β,10β,17β-trihydroxy-19-nor-4-androsten-3-one (4), along with four known metabolites, 10β,17β-dihydroxy-19-nor-4-androsten-3-one (5), 6β,17β-dihydroxy-19-nor-4-androsten-3-one (6) 10β-hydroxy-19-nor-4-androsten-3,17-dione (7) and 16β,17β-dihydroxy-19-nor-4-androsten-3-one (8). Compounds 1-8 were evaluated for their anti-leishmanial activity. Compounds 1 and 8 showed a significant activity in vitro against Leishmania major. The leishmanicidal potential of compounds 1-8 (IC50=32.0±0.5, >100, 77.39±5.52, 70.90±1.16, 54.94±1.01, 80.23±3.39, 61.12±1.39 and 29.55±1.14 μM, respectively) can form the basis for the development of effective therapies against the protozoal tropical disease leishmaniasis.

Ultraviolet irradiation produces novel endonuclease III-sensitive cytosine photoproducts at dipyrimidine sites

Ultraviolet light irradiation of DNA in vitro and in vivo induces cyclobutane dimers, (6–4) pyrimidine‐pyrimi‐done photoproducts and a variety of minor products. Using a denned DNA fragment, we have identified two classes of sites that can be cleaved by Escherichia coli endonuclease III: single cytokines whose heat lability corresponds to that of cytosine hydrates and more heat‐stable dipyrimidines containing cytosine. The dipyrimidine products are induced at sites suggestive of (6–4) photoproducts but are not recognized as (6–4) photoproducts by radioimmunoassay. Use of oligonucleotides containing a single cyclobutane thymine dimer, a (6–4) photoproduct or the Dewar photoisomer of the (6–4) photoproduct also indicated that these products are not substrates for endonuclease III. We have therefore identified a minor UV photoproduct that has the same sequence specificity as the two major dipyrimidine photoproducts; it may be a minor isomer, a unique derivative or an oxidative lesion confined to dipyrimidine sites. Its biological significance is not yet known but may be masked by the preponderance of major products at the same sites. Its occurrence at the particular site in dipyrimidine sequences involved in the mutagenic action of UV photoproducts suggests that it may play a role in generating C to T transitions that are common UV‐induced mutations.

The radiosensitizer 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide induces DNA damage in EMT-6 mammary carcinoma cells

BackgroundDCQ (2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide), a synthetic quinoxaline 1,4-dioxide, enhances the cytotoxic effect of ionizing radiation (IR) in vivo and in vitro. We sought to clarify whether increased radiation-induced DNA damage, decreased rate of damage repair, and the generation of reactive oxygen species (ROS) contribute to DCQ enhancement of IR.MethodsMurine mammary adenocarcinoma EMT-6 cells were treated with DCQ for 4 h before exposure to 10 Gy IR. Treated cells were monitored for modulations in cell cycle, induction of DNA damage, and generation of ROS.ResultsCombined DCQ and IR treatments (DCQ+IR) induced rapid cell-cycle arrests in EMT-6 cells, particularly in S and G2/M phases. Alkaline comet assays revealed high levels of DNA damage in cells after exposure to DCQ+IR, consistent with damage-induced arrest. Unlike IR-only and DCQ-only treated cells, the damage induced by combined DCQ+IR was repaired at a slower rate. Combined treatment, compared to separate DCQ and IR treatments, activated DNA-protein kinase and induced more p-ATM, supporting a role for double strand breaks (DSBs), which are more toxic and difficult to repair than single strand breaks (SSBs). Contributing factors to DCQ radiosensitization appear to be the induction of ROS and DSBs.ConclusionCollectively, our findings indicate that radiosensitization by DCQ is mediated by DNA damage and decreased repair and that ROS are at least partially responsible.

Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

Transcription antitermination in phages lambda and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the lambda N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB(left) complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmid-based antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and lambda N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by lambda N. Point mutations can dramatically alter boxB specificity between P22 and lambda N. A boxB specific for P22 N can be mutated to lambda N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.

Terellia fuscicornis (Diptera: Tephritidae): biological and morphological adaptation on artichoke and milk thistle

The biology, morphology, and genetics of Terellia fuscicornis on cultivated artichoke and its new recorded host, Silybum marianum, are examined. Morphometrically, the two host‐associated populations were distinct. Except for egg length, all immature stages of the artichoke‐associated population were larger. Principal component analysis, based on head and wing measurements, showed that the adults from both populations clustered into two groups, with the adults reared from artichoke being larger. The ovipositor was significantly longer and blunter in the artichoke‐associated females, reflecting the larger flower heads exploited. Male terminalia measurements showed significant differences between the two populations. Several differences in courtship behaviour were detected between the two populations. Although the two populations show allochronic isolation, sequencing of two mitochondrial genes, ND1 and COI, and one nuclear gene, 18S, revealed no genetic differences between the two populations.

Host races in Chaetostomella cylindrica (Diptera: Tephritidae): genetic and behavioural evidence.

The highly oligophagous tephritid Chaetostomella cylindrica infests the flower heads of six genera and ten species of thistles in Lebanon. It predominantly utilizes two hosts occurring in sympatry, Notobasis syriaca and Onopordum illyricum. Previous work showed that adult flies emerging from N. syriaca fit more closely the description of the species, particularly with respect to the colour and pattern on the mesonotum; furthermore, significant differences were observed between the aculeus shape and length. This study investigates the biology of the immatures and compares adults from the two host races behaviourally and genetically. Larvae of both races fed in a similar way, with each larva destroying 3-10 achenes; however, the oviposition behaviour of females differed. Females of the Onopordum-associated flies laid an average of three eggs per head, and deposited the eggs glued to each others in a cluster, while females of the Notobasis-associated flies deposited their eggs unattached, usually with one egg per head. Subtle differences were also observed in the post-mating behaviour of adult males. DNA sequencing of an amplified fragment of the mitochondrial NADH-dehydrogenase subunit 1 gene revealed 44 single nucleotide polymorphisms in 622 base pairs. A PCR-RFLP method was developed to distinguish the two host-associated populations. Together with previously published morphometric studies, our data show that C. cylindrica consists of distinct host races, which seem to be reproductively isolated as two separate genetic lineages were observed.