Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

STRUCTURAL ANALYSIS OF THE TRANSCRIPTION PRODUCTS OF A TEMPERATURE-SENSITIVE PHENOTYPIC MUTANT OF MOLONEY SARCOMA VIRUS--MUSV TS110

Cells infected with a temperature sensitive phenotypic mutant of Moloney sarcoma virus (MuSVts110) exhibit a transformed phenotype at 33('(DEGREES)) and synthesize two virus specific proteins, p85('gag-mos), a gag-mos fusion protein and p58('gag), a truncated gag precursor protein (the gag gene codes for viral structural proteins and mos is the MuSV transforming gene). At 39('(DEGREES)) only p58('gag) is synthesized and the morphology of the cells is similar to uninfected NRK parental cells. Two MuSVts110 specific RNAs are made in MuSVts110-infected cells, one of 4.0 kb in length, the other of 3.5 kb. Previous work indicated that each of these RNAs arose by a single central deletion of parental MuSV genetic material, and that p58('gag) was made by the 4.0 kb RNA and p85('gag-mos) from the 3.5 kb RNA. The objective of my dissertation research was to map precisely the deletion boundaries of both of the MuSVts110 RNAs, and to determine the proper reading frame across both deletion borders. This work succeeded in arriving at the following conclusions: (a) Using S-1 nuclease analysis and primer extension sequencing, it was found that the 4.0 kb MuSVts110 RNA arose by a 1488 base deletion of 5.2 kb parental MuSV genomic RNA. This deletion resulted in an out of frame fusion of the gag and mos genes that resulted in the formation of a "stop" codon which causes termination of translation just beyond the c-terminus of the gag region. Thus, this RNA can only be translated into the truncated gag protein p58('gag). (b) S-1 analysis of RNA from cells cultivated at different temperatures demonstrated that the 4.0 kb RNA was synthesized at all temperatures but that synthesis of the 3.5 kb RNA was temperature sensitive. These observations supported the data derived from blot hybridization experiments the interpretation of which argued for the existence of a single provirus in MuSVts110 infected cells, and hence only a single primary transcript (the 4.0 kb RNA). (c) Analyses similar to those described in (a) above showed that the 3.5 kb RNA was derived from the 4.0 kb MuSVts110 RNA by a further deletion of 431 bases, fusing the gag and mos genes into a continuous reading frame capable of directing synthesis of the p85('gag-mos) protein. These sequence data and the presence of only one MuSVts110-specific provirus, indicate that a splice mechanism is employed to generate the 3.5 kb RNA since the gag and mos genes are observed to be fused in frame in this RNA. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

RNA binding characteristics and overall topology of the vaccinia virus polyadenylation complex

mRNA 3′ polyadenylation is central to mRNA biogenesis in prokaryotes and eukaryotes, and is implicated in numerous aspects of mRNA metabolism, including efficiency of mRNA export from the nucleus, message stability, and initiation of translation. However, due to the great complexity of the eukaryotic polyadenylation apparatus, the mechanisms of RNA 3 ′ end processing have remained elusive. Although the RNA processing reactions leading to polyadenylated messenger RNA have been studied in many systems, and much progress has been made, a complete understanding of the biochemistry of the poly(A) polymerase enzyme is still lacking. My research uses Vaccinia virus as a model system to gain a better understanding of this complicated polyadenylation process, which consist of RNA binding, catalysis and polymerase translocation. ^ Vaccinia virus replicates in the cytoplasm of its host cell, so it must employ its own poly(A) polymerase (PAP), a heterodimer of two virus encoded proteins, VP55 and VP39. VP55 is the catalytic subunit, adding 30 adenylates to a non-polyadenylated RNA in a rapid processive manner before abruptly changing to a slow, non-processive mode of adenylate addition and dissociating from the RNA. VP39 is the stimulatory subunit. It has no polyadenylation catalytic activity by itself, but when associated with VP55 it facilitates the semi-processive synthesis of tails several hundred adenylates in length. ^ Oligonucleotide selection and competition studies have shown that the heterodimer binds a minimal motif of (rU)2 (N)25 U, the “heterodimer binding motif”, within an oligonucleotide, and its primer selection for polyadenylation is base-type specific. ^ Crosslinking studies using photosensitive uridylate analogs show that within a VP55-VP39-primer ternary complex, VP55 comes into contact with all three required uridylates, while VP39 only contacts the downstream uridylate. Further studies, using a backbone-anchored photosensitive crosslinker show that both PAP subunits are in close proximity to the downstream −10 to −21 region of 50mer model primers containing the heterodimer binding motif. This equal crosslinking to both subunits suggests that the dimerization of VP55 and VP39 creates either a cleft or a channel between the two subunits through which this region of RNA passes. ^ Peptide mapping studies of VP39 covalently crosslinked to the oligonucleotide have identified residue R107 as the amino acid in close proximity to the −10 uridylate. This helps us project a conceptual model onto the known physical surface of this subunit. In the absence of any tertiary structural data for VP55, we have used a series of oligonucleotide selection assays, as well as crosslinking, nucleotide transfer assays, and gel shift assays to gain insight into the requirements for binding, polyadenylation and translocation. Collectively, these data allow us to put together a comprehensive model of the structure and function of the polyadenylation ternary complex consisting of VP39, VP55 and RNA. ^

Identification and characterization of distinct classes of cAMP-pulse-induced genes in Dictyostelium discoideum development

The unicellular amoeba Dictyostelium discoideum embarks on a developmental program upon starvation. During development, extracellular oscillatory cAMP signaling orchestrates the chemotaxis-mediated aggregation of ∼105 amoebae and is required for optimal induction of so-called pulse-induced genes. This requirement for pulsatile CAMP reflects adaptation of the cAMP-receptor-mediated pathways that regulate these genes. Through examination of a collection of pulse-induced genes, we defined two distinct gene classes based on their induction kinetics and the impact of mutations that impair PKA signaling. The first class (represented by D2 and prtA) is highly dependent on PKA signaling, whereas the second class (represented by carA, gpaB, and acaA) is not. Analysis of expression kinetics revealed that these classes are sequentially expressed with the PKA-independent genes peaking in expression before the PKA-dependent class. Experiments with cycloheximide, an inhibitor of translation, demonstrated that the pulse induction of both classes depends on new protein synthesis early in development. carA and gpaB also exhibit pulse-independent, starvation-induced expression which, unlike their pulse induction, was found to be insensitive to cycloheximide added at the outset of starvation. This result indicates that the mechanism of starvation induction pre-exists in growing cells and is distinct from the pulse induction mechanism for these genes. In order to identify cis-acting elements that are critical for induction of carA, we constructed a GFP reporter controlled by a 914-base-pair portion of its promoter and verified that its expression was PKA-independent, pulse-inducible, and developmentally regulated like the endogenous carA gene. By a combination of truncation, internal deletion, and site-directed mutation, we defined several distinct functional elements within the carA promoter, including a 39-bp region required for pulse induction between base pairs -321 and -282 (relative to the transcription start site), a 131-bp region proximal to the start site that is sufficient for starvation induction, and two separate enhancer domains. Identification of factors that interact with these promoter elements and genetic approaches exploiting the GFP reporter described here should help complete our understanding of the mechanisms regulating these genes, including adaptation mechanisms that likely also govern chemotaxis of Dictyostelium and mammalian cells. ^

Mechanism of translationally coupled mRNA turnover mediated by c-fos protein-coding-region determinant

Proto-oncogene c-fos is a member of the class of early-response genes whose transient expression plays a crucial role in cell proliferation, differentiation, and apoptosis. Degradation of c- fos mRNA is an important mechanism for controlling c-fos expression. Rapid mRNA turnover mediated by the protein-coding-region determinant (mCRD) of the c-fos transcript illustrates a functional interplay between mRNA turnover and translation that coordinately influences the fate of cytoplasmic mRNA. It is suggested that mCRD communicates with the 3′ poly(A) tail via an mRNP complex comprising mCRD-associated proteins, which prevents deadenylation in the absence of translation. Ribosome transit as a result of translation is required to alter the conformation of the mRNP complex, thereby eliciting accelerated deadenylation and mRNA decay. To gain further insight into the mechanism of mCRD-mediated mRNA turnover, Unr was identified as an mCRD-binding protein, and its binding site within mCRD was characterized. Moreover, the functional role for Unr in mRNA decay was demonstrated. The result showed that elevation of Unr protein level in the cytoplasm led to inhibition of mRNA destabilization by mCRD. In addition, GST pull-down assay and immuno-precipitation analysis revealed that Unr interacted with PABP in an RNA-independent manner, which identified Unr as a novel PABP-interacting protein. Furthermore, the Unr interacting domain in PABP was characterized. In vivo mRNA decay experiments demonstrated a role for Unr-PABP interaction in mCRD-mediated mRNA decay. In conclusion, the findings of this study provide the first evidence that Unr plays a key role in mCRD-mediated mRNA decay. It is proposed that Unr is recruited by mCRD to initiate the formation of a dynamic mRNP complex for communicating with poly(A) tail through PABP. This unique mRNP complex may couple translation to mRNA decay, and perhaps to recruit the responsible nuclease for deadenylation. ^

An automatic translation scheme from prolog to the andorra kernel language

The Andorra family of languages (which includes the Andorra Kernel Language -AKL) is aimed, in principie, at simultaneously supporting the programming styles of Prolog and committed choice languages. On the other hand, AKL requires a somewhat detailed specification of control by the user. This could be avoided by programming in Prolog to run on AKL. However, Prolog programs cannot be executed directly on AKL. This is due to a number of factors, from more or less trivial syntactic differences to more involved issues such as the treatment of cut and making the exploitation of certain types of parallelism possible. This paper provides basic guidelines for constructing an automatic compiler of Prolog programs into AKL, which can bridge those differences. In addition to supporting Prolog, our style of translation achieves independent and-parallel execution where possible, which is relevant since this type of parallel execution preserves, through the translation, the user-perceived "complexity" of the original Prolog program.

An automatic translation scheme from CLP to AKL

The Andorra Kernel language scheme was aimed, in principle, at simultaneously supporting the programming styles of Prolog and committed choice languages. Within the constraint programming paradigm, this family of languages could also in principle support the concurrent constraint paradigm. This happens for the Agents Kernel Language (AKL). On the other hand, AKL requires a somewhat detailed specification of control by the user. This could be avoided by programming in CLP to run on AKL. However, CLP programs cannot be executed directly on AKL. This is due to a number of factors, from more or less trivial syntactic differences to more involved issues such as the treatment of cut and making the exploitation of certain types of parallelism possible. This paper provides a translation scheme which is a basis of an automatic compiler of CLP programs into AKL, which can bridge those differences. In addition to supporting CLP, our style of translation achieves independent and-parallel execution where possible, which is relevant since this type of parallel execution preserves, through the translation, the user-perceived "complexity" of the original program.

New methods for the synthesis of radiation patterns of coupled antenna arrays = Nuevos métodos de síntesis de diagramas de radiación de agrupaciones de antenas acopladas

El principal objetivo de esta tesis es el desarrollo de métodos de síntesis de diagramas de radiación de agrupaciones de antenas, en donde se realiza una caracterización electromagnética rigurosa de los elementos radiantes y de los acoplos mutuos existentes. Esta caracterización no se realiza habitualmente en la gran mayoría de métodos de síntesis encontrados en la literatura, debido fundamentalmente a dos razones. Por un lado, se considera que el diagrama de radiación de un array de antenas se puede aproximar con el factor de array que únicamente tiene en cuenta la posición de los elementos y las excitaciones aplicadas a los mismos. Sin embargo, como se mostrará en esta tesis, en múltiples ocasiones un riguroso análisis de los elementos radiantes y del acoplo mutuo entre ellos es importante ya que los resultados obtenidos pueden ser notablemente diferentes. Por otro lado, no es sencillo combinar un método de análisis electromagnético con un proceso de síntesis de diagramas de radiación. Los métodos de análisis de agrupaciones de antenas suelen ser costosos computacionalmente, ya que son estructuras grandes en términos de longitudes de onda. Generalmente, un diseño de un problema electromagnético suele comprender varios análisis de la estructura, dependiendo de las variaciones de las características, lo que hace este proceso muy costoso. Dos métodos se utilizan en esta tesis para el análisis de los arrays acoplados. Ambos están basados en el método de los elementos finitos, la descomposición de dominio y el análisis modal para analizar la estructura radiante y han sido desarrollados en el grupo de investigación donde se engloba esta tesis. El primero de ellos es una técnica de análisis de arrays finitos basado en la aproximación de array infinito. Su uso es indicado para arrays planos de grandes dimensiones con elementos equiespaciados. El segundo caracteriza el array y el acoplo mutuo entre elementos a partir de una expansión en modos esféricos del campo radiado por cada uno de los elementos. Este método calcula los acoplos entre los diferentes elementos del array usando las propiedades de traslación y rotación de los modos esféricos. Es capaz de analizar agrupaciones de elementos distribuidos de forma arbitraria. Ambas técnicas utilizan una formulación matricial que caracteriza de forma rigurosa el campo radiado por el array. Esto las hace muy apropiadas para su posterior uso en una herramienta de diseño, como los métodos de síntesis desarrollados en esta tesis. Los resultados obtenidos por estas técnicas de síntesis, que incluyen métodos rigurosos de análisis, son consecuentemente más precisos. La síntesis de arrays consiste en modificar uno o varios parámetros de las agrupaciones de antenas buscando unas determinadas especificaciones de las características de radiación. Los parámetros utilizados como variables de optimización pueden ser varios. Los más utilizados son las excitaciones aplicadas a los elementos, pero también es posible modificar otros parámetros de diseño como son las posiciones de los elementos o las rotaciones de estos. Los objetivos de las síntesis pueden ser dirigir el haz o haces en una determinada dirección o conformar el haz con formas arbitrarias. Además, es posible minimizar el nivel de los lóbulos secundarios o del rizado en las regiones deseadas, imponer nulos que evitan posibles interferencias o reducir el nivel de la componente contrapolar. El método para el análisis de arrays finitos basado en la aproximación de array infinito considera un array finito como un array infinito con un número finito de elementos excitados. Los elementos no excitados están físicamente presentes y pueden presentar tres diferentes terminaciones, corto-circuito, circuito abierto y adaptados. Cada una de estas terminaciones simulará mejor el entorno real en el que el array se encuentre. Este método de análisis se integra en la tesis con dos métodos diferentes de síntesis de diagramas de radiación. En el primero de ellos se presenta un método basado en programación lineal en donde es posible dirigir el haz o haces, en la dirección deseada, además de ejercer un control sobre los lóbulos secundarios o imponer nulos. Este método es muy eficiente y obtiene soluciones óptimas. El mismo método de análisis es también aplicado a un método de conformación de haz, en donde un problema originalmente no convexo (y de difícil solución) es transformado en un problema convexo imponiendo restricciones de simetría, resolviendo de este modo eficientemente un problema complejo. Con este método es posible diseñar diagramas de radiación con haces de forma arbitraria, ejerciendo un control en el rizado del lóbulo principal, así como en el nivel de los lóbulos secundarios. El método de análisis de arrays basado en la expansión en modos esféricos se integra en la tesis con tres técnicas de síntesis de diagramas de radiación. Se propone inicialmente una síntesis de conformación del haz basado en el método de la recuperación de fase resuelta de forma iterativa mediante métodos convexos, en donde relajando las restricciones del problema original se consiguen unas soluciones cercanas a las óptimas de manera eficiente. Dos métodos de síntesis se han propuesto, donde las variables de optimización son las posiciones y las rotaciones de los elementos respectivamente. Se define una función de coste basada en la intensidad de radiación, la cual es minimizada de forma iterativa con el método del gradiente. Ambos métodos reducen el nivel de los lóbulos secundarios minimizando una función de coste. El gradiente de la función de coste es obtenido en términos de la variable de optimización en cada método. Esta función de coste está formada por la expresión rigurosa de la intensidad de radiación y por una función de peso definida por el usuario para imponer prioridades sobre las diferentes regiones de radiación, si así se desea. Por último, se presenta un método en el cual, mediante técnicas de programación entera, se buscan las fases discretas que generan un diagrama de radiación lo más cercano posible al deseado. Con este método se obtienen diseños que minimizan el coste de fabricación. En cada uno de las diferentes técnicas propuestas en la tesis, se presentan resultados con elementos reales que muestran las capacidades y posibilidades que los métodos ofrecen. Se comparan los resultados con otros métodos disponibles en la literatura. Se muestra la importancia de tener en cuenta los diagramas de los elementos reales y los acoplos mutuos en el proceso de síntesis y se comparan los resultados obtenidos con herramientas de software comerciales. ABSTRACT The main objective of this thesis is the development of optimization methods for the radiation pattern synthesis of array antennas in which a rigorous electromagnetic characterization of the radiators and the mutual coupling between them is performed. The electromagnetic characterization is usually overlooked in most of the available synthesis methods in the literature, this is mainly due to two reasons. On the one hand, it is argued that the radiation pattern of an array is mainly influenced by the array factor and that the mutual coupling plays a minor role. As it is shown in this thesis, the mutual coupling and the rigorous characterization of the array antenna influences significantly in the array performance and its computation leads to differences in the results obtained. On the other hand, it is difficult to introduce an analysis procedure into a synthesis technique. The analysis of array antennas is generally expensive computationally as the structure to analyze is large in terms of wavelengths. A synthesis method requires to carry out a large number of analysis, this makes the synthesis problem very expensive computationally or intractable in some cases. Two methods have been used in this thesis for the analysis of coupled antenna arrays, both of them have been developed in the research group in which this thesis is involved. They are based on the finite element method (FEM), the domain decomposition and the modal analysis. The first one obtains a finite array characterization with the results obtained from the infinite array approach. It is specially indicated for the analysis of large arrays with equispaced elements. The second one characterizes the array elements and the mutual coupling between them with a spherical wave expansion of the radiated field by each element. The mutual coupling is computed using the properties of translation and rotation of spherical waves. This method is able to analyze arrays with elements placed on an arbitrary distribution. Both techniques provide a matrix formulation that makes them very suitable for being integrated in synthesis techniques, the results obtained from these synthesis methods will be very accurate. The array synthesis stands for the modification of one or several array parameters looking for some desired specifications of the radiation pattern. The array parameters used as optimization variables are usually the excitation weights applied to the array elements, but some other array characteristics can be used as well, such as the array elements positions or rotations. The desired specifications may be to steer the beam towards any specific direction or to generate shaped beams with arbitrary geometry. Further characteristics can be handled as well, such as minimize the side lobe level in some other radiating regions, to minimize the ripple of the shaped beam, to take control over the cross-polar component or to impose nulls on the radiation pattern to avoid possible interferences from specific directions. The analysis method based on the infinite array approach considers an infinite array with a finite number of excited elements. The infinite non-excited elements are physically present and may have three different terminations, short-circuit, open circuit and match terminated. Each of this terminations is a better simulation for the real environment of the array. This method is used in this thesis for the development of two synthesis methods. In the first one, a multi-objective radiation pattern synthesis is presented, in which it is possible to steer the beam or beams in desired directions, minimizing the side lobe level and with the possibility of imposing nulls in the radiation pattern. This method is very efficient and obtains optimal solutions as it is based on convex programming. The same analysis method is used in a shaped beam technique in which an originally non-convex problem is transformed into a convex one applying symmetry restrictions, thus solving a complex problem in an efficient way. This method allows the synthesis of shaped beam radiation patterns controlling the ripple in the mainlobe and the side lobe level. The analysis method based on the spherical wave expansion is applied for different synthesis techniques of the radiation pattern of coupled arrays. A shaped beam synthesis is presented, in which a convex formulation is proposed based on the phase retrieval method. In this technique, an originally non-convex problem is solved using a relaxation and solving a convex problems iteratively. Two methods are proposed based on the gradient method. A cost function is defined involving the radiation intensity of the coupled array and a weighting function that provides more degrees of freedom to the designer. The gradient of the cost function is computed with respect to the positions in one of them and the rotations of the elements in the second one. The elements are moved or rotated iteratively following the results of the gradient. A highly non-convex problem is solved very efficiently, obtaining very good results that are dependent on the starting point. Finally, an optimization method is presented where discrete digital phases are synthesized providing a radiation pattern as close as possible to the desired one. The problem is solved using linear integer programming procedures obtaining array designs that greatly reduce the fabrication costs. Results are provided for every method showing the capabilities that the above mentioned methods offer. The results obtained are compared with available methods in the literature. The importance of introducing a rigorous analysis into the synthesis method is emphasized and the results obtained are compared with a commercial software, showing good agreement.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA′-′lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

The p20 and Ded1 proteins have antagonistic roles in eIF4E-dependent translation in Saccharomyces cerevisiae

The translation initiation factor eIF4E mediates the binding of the small ribosomal subunit to the cap structure at the 5′ end of the mRNA. In Saccharomyces cerevisiae, the cap-binding protein eIF4E is mainly associated with eIF4G, forming the cap-binding complex eIF4F. Other proteins are detected upon purification of the complex on cap-affinity columns. Among them is p20, a protein of unknown function encoded by the CAF20 gene. Here, we show a negative regulatory role for the p20 protein in translation initiation. Deletion of CAF20 partially suppresses mutations in translation initiation factors. Overexpression of the p20 protein results in a synthetic enhancement of translation mutation phenotypes. Similar effects are observed for mutations in the DED1 gene, which we have isolated as a multicopy suppressor of a temperature-sensitive eIF4E mutation. The DED1 gene encodes a putative RNA helicase of the DEAD-box family. The analyses of its suppressor activity, of polysome profiles of ded1 mutant strains, and of synthetic lethal interactions with different translation mutants indicate that the Ded1 protein has a role in translation initiation in S. cerevisiae.

Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids.

Endoplasmic Reticulum Stress-induced mRNA Splicing Permits Synthesis of Transcription Factor Hac1p/Ern4p That Activates the Unfolded Protein Response

An intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, called the unfolded protein response (UPR), is activated when unfolded proteins are accumulated in the ER under a variety of stress conditions (“ER stress”). We and others recently identified Hac1p/Ern4p as a transcription factor responsible for the UPR in Saccharomyces cerevisiae. It was further reported that Hac1p (238 aa) is detected only in ER-stressed cells, and its expression is mediated by unconventional splicing of HAC1 precursor mRNA. The splicing replaces the C-terminal portion of Hac1p; it was proposed that precursor mRNA is also translated but the putative product of 230 aa is rapidly degraded by the ubiquitin–proteasome pathway. We have identified and characterized the same regulated splicing and confirmed its essential features. Contrary to the above proposal, however, we find that the 238-aa product of mature mRNA and the 230-aa-type protein tested are highly unstable with little or no difference in stability. Furthermore, we demonstrate that the absence of Hac1p in unstressed cells is due to the lack of translation of precursor mRNA. We conclude that Hac1p is synthesized as the result of ER stress-induced mRNA splicing, leading to activation of the UPR.

Solution structure of the A loop of 23S ribosomal RNA

The A loop is an essential RNA component of the ribosome peptidyltransferase center that directly interacts with aminoacyl (A)-site tRNA. The A loop is highly conserved and contains a ubiquitous 2′-O-methyl ribose modification at position U2552. Here, we present the solution structure of a modified and unmodified A-loop RNA to define both the A-loop fold and the structural impact of the U2552 modification. Solution data reveal that the A-loop RNA has a compact structure that includes a noncanonical base pair between C2556 and U2552. NMR evidence is presented that the N3 position of C2556 has a shifted pKa and that protonation at C2556-N3 changes the C-U pair geometry. Our data indicate that U2552 methylation modifies the A-loop fold, in particular the dynamics and position of residues C2556 and U2555. We compare our structural data with the structure of the A loop observed in a recent 50S crystal structure [Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000) Science 289, 905–920; Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. (2000) Science 289, 920–930]. The solution and crystal structures of the A loop are dramatically different, suggesting that a structural rearrangement of the A loop must occur on docking into the peptidyltransferase center. Possible roles of this docking event, the shifted pKa of C2556 and the U2552 2′-O-methylation in the mechanism of translation, are discussed.

Characterization of Mutants with Alterations of the Phosphorylation Site in the D2 Photosystem II Polypeptide of Chlamydomonas reinhardtii1

We have changed the potential phosphorylation site, a threonine residue at position 2 of the D2 polypeptide of the photosystem II complex of Chlamydomonas reinhardtii, to alanine, valine, aspartate, proline, glycine, or glutamate. Mutants with neutral amino acid changes did not display any phenotype with regard to photoautotrophic growth, light sensitivity, fluorescence transients, or photoinhibition. Pulse labeling of these mutants with 32P indicated that a phosphorylated protein of the same size as D2 is absent in these mutants, suggesting that threonine-2 is indeed the unique phosphorylation site of D2. In contrast, mutants in which threonine-2 has been replaced with acidic residues are deficient in photosystem II. Use of chimeric genes containing the psbD 5′-untranslated region revealed that the initiation of translation was not affected in these mutants, but the mutations interfered with a later step of D2 synthesis and accumulation.