Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.     

Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda S

lambda S, the prototype class I holin gene, encodes three potential transmembrane domains in its 107 codons, whereas 21 S, the class II prototype spans only 71 codons and encodes two transmembrane domains. Many holin genes, including lambda S and 21 S, have the "dual-start" regulatory motif at the N terminus, suggesting that class I and II holins have the same topology. The primary structure of 21 S strongly suggests a bitopic "helical-hairpin" topology, with N and C termini on the cytoplasmic side of the membrane. However, lambda S chimeras with an N-terminal signal sequence show Lep-dependent function, indicating that the N-terminal domain of S requires export. Here the signal sequence chimera is shown to be sensitive to the missense change A52V, which blocks normal S function. Moreover, cysteine-modification studies in isolated membranes using a collection of S variants with single-cysteine substitutions show that the positions in the core of the 3 putative transmembrane domains of lambda S are protected. Also, S proteins with single-cysteine substitutions in the predicted cytoplasmic and periplasmic loops are more efficiently labeled in inverted membrane vesicles and whole cells, respectively. These data constitute direct evidence that the holin S(lambda) has three transmembrane domains and indicate that class I and class II holins have different topologies, despite regulatory and functional homology.     

Non-canonical mechanism for translational control in bacteria: synthesis of ribosomal protein S1

Translation initiation region (TIR) of the rpsA mRNA encoding ribosomal protein S1 is one of the most efficient in Escherichia coli despite the absence of a canonical Shine-Dalgarno-element. Its high efficiency is under strong negative autogenous control, a puzzling phenomenon as S1 has no strict sequence specificity. To define sequence and structural elements responsible for translational efficiency and autoregulation of the rpsA mRNA, a series of rpsA'-'lacZ chromosomal fusions bearing various mutations in the rpsA TIR was created and tested for beta-galactosidase activity in the absence and presence of excess S1. These in vivo results, as well as data obtained by in vitro techniques and phylogenetic comparison, allow us to propose a model for the structural and functional organization of the rpsA TIR specific for proteobacteria related to E.coli. According to the model, the high efficiency of translation initiation is provided by a specific fold of the rpsA leader forming a non-contiguous ribosome entry site, which is destroyed upon binding of free S1 when it acts as an autogenous repressor.     

Physical evidence for distinct mechanisms of translational control by upstream open reading frames.

The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream open reading frames (uORFs) and the CPA1 leader contains a single uORF. To determine how these uORFs control translation, we examined mRNAs containing these leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translation. Ribosomes predominantly loaded first at GCN4 uORF1. Following its translation, but not the translation of uORF4, they efficiently reinitiated protein synthesis at Gcn4p. Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p. This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro. In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstream start codon by scanning past the uORF. Addition of arginine caused ribosomes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocking scanning ribosomes, and not by affecting reinitiation. The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways.     

Dominance of mutations affecting viability in Drosophila melanogaster

There have been several attempts to estimate the average dominance (ratio of heterozygous to homozygous effects) of spontaneous deleterious mutations in Drosophila melanogaster, but these have given inconsistent results. We investigated whether transposable element (TE) insertions have higher average dominance for egg-to-adult viability than do point mutations, a possibility suggested by the types of fitness-depressing effects that TEs are believed to have. If so, then variation in dominance estimates among strains and crosses would be expected as a consequence of variation in TE activity. As a first test, we estimated the average dominance of all mutations and of copia insertions in a set of lines that had accumulated spontaneous mutations for 33 generations. A traditional regression method gave a dominance estimate for all mutations of 0.17, whereas average dominance of copia insertions was 0.51; the difference between these two estimates approached significance (P = 0.08). As a second test, we reanalyzed Ohnishi 1974 data on dominance of spontaneous and EMS-induced mutations. Because a considerable fraction of spontaneous mutations are caused by TE insertions, whereas EMS induces mainly point mutations, we predicted that average dominance would decline with increasing EMS concentration. This pattern was observed, but again fell short of formal significance (P = 0.07). Taken together, however, the two results give modest support for the hypothesis that TE insertions have greater average dominance in their viability effects than do point mutations, possibly as a result of deleterious effects of expression of TE-encoded genes.     

Isocitrate dehydrogenase mutations: new opportunities for translational research

Over the last decade, comprehensive genome-wide sequencing studies have enabled us to find out unexpected genetic alterations of metabolism in cancer. An example is the identification of arginine missense mutations of isocitrate dehydrogenases-1 and -2 (IDH1/2) in glioma, acute myeloid leukemia (AML), chondrosarcomas, and cholangiocarcinoma. These alterations are closely associated with the production of a new stereospecific metabolite, (R)-2-hydroxyglutarate (R-2HG). A large number of follow-up studies have been performed to address the molecular mechanisms of IDH1/2 mutations underlying how these events contribute to malignant transformation. In the meanwhile, the development of selective mutant IDH1/2 chemical inhibitors is being actively pursued in the scientific community and pharmaceutical industry. The present review article briefly discusses the important findings that highlight the molecular mechanisms of IDH1/2 mutations in cancer and provides a current status for development of selective mutant IDH1/2 chemical inhibitors. [BMB Reports 2015; 48(5): 266-270]     

Dominance and overdominance of mildly deleterious induced mutations for fitness traits in Caenorhabditis elegans

We estimated the average dominance coefficient of mildly deleterious mutations (h, the proportion by which mutations in the heterozygous state reduce fitness components relative to those in the homozygous state) in the nematode Caenorhabditis elegans. From 56 worm lines that carry mutations induced by the point mutagen ethyl methanesulfonate (EMS), we selected 19 lines that are relatively high in fitness and estimated the viabilities, productivities, and relative fitnesses of heterozygotes and homozygotes compared to the ancestral wild type. There was very little effect of homozygous or heterozygous mutations on egg-to-adult viability. For productivity and relative fitness, we found that the average dominance coefficient, h, was approximately 0.1, suggesting that mildly deleterious mutations are on average partially recessive. These estimates were not significantly different from zero (complete recessivity) but were significantly different from 0.5 (additivity). In addition, there was a significant amount of variation in h among lines, and analysis of average dominance coefficients of individual lines suggested that several lines showed overdominance for fitness. Further investigation of two of these lines partially confirmed this finding.     

Cytoplasmic polyadenylation and translational control

Cytoplasmic polyadenylation is the process by which dormant, translationally inactive mRNAs become activated via the elongation of their poly(A) tails in the cytoplasm. This process is regulated by the conserved cytoplasmic polyadenylation element binding (CPEB) protein family. Recent studies have advanced our understanding of the molecular code that dictates the timing of CPEB-mediated poly(A) tail elongation and the extent of translational activation. In addition, evidence for CPEB-independent mechanisms has accumulated, and the breath of biological circumstances in which cytoplasmic polyadenylation plays a role has expanded. These observations underscore the versatility of CPEB as a translational regulator, and highlight the diversity of cytoplasmic polyadenylation mechanisms.     

Readthrough compounds for nonsense mutations: bridging the translational gap

Approximately 10% of all pathological mutations are nonsense mutations that are responsible for several severe genetic diseases for which no treatment regimens are currently available. The most widespread strategy for treating nonsense mutations is by enhancing ribosomal readthrough of premature termination codons (PTCs) to restore the production of the full-length protein. In the past decade several compounds with readthrough potential have been identified. However, although preclinical results on these compounds are promising, clinical studies have not yielded positive outcomes. We review preclinical and clinical research related to readthrough compounds and characterize factors that contribute to the observed translational gap.     

The V lambda J lambda repertoire in human fetal spleen: evidence for positive selection and extensive receptor editing

VlambdaJlambda rearrangements obtained from genomic DNA of individual IgM(+) B cells from human fetal spleen were analyzed. A nonrandom pattern of lambda gene rearrangements that differed from the adult Vlambda repertoire was found. The Vlambda distal genes 8A and 4B were absent from the nonproductive fetal repertoire, whereas 2E and 3L were overrepresented and 1B was underrepresented in the productive fetal repertoire. Positive selection of the Vlambda gene, 2E, along with Vlambda rearrangements employing homologous VlambdaJlambda joins were observed in the fetal, but not in the adult Vlambda repertoire. Overrepresentation of Jlambda distal cluster C genes rearranging to the Vlambda distal J segment, Jlambda7, in both productive and nonproductive fetal repertoires suggested that receptor editing/replacement was more active in the fetus than in adults. Numerous identical VlambdaJlambda junctions were observed in both the productive and nonproductive repertoire of the fetus and adult, but were significantly more frequent in the productive repertoire of the fetus, suggesting expansion of B cells expressing particular lambda-light chains in both stages of development, with more profound expansion in the fetal repertoire. Notably, B cells expressing identical lambda-light chains expressed diverse heavy chains. These data demonstrate that three mechanisms strongly influence the shaping of the human fetal lambda-chain repertoire that are less evident in the adult: positive selection, receptor editing, and expansion of B cells expressing specific lambda-light chains. These events imply that the expressed fetal repertoire is shaped by exposure to self Ags.     

A combinatorial code for CPE-mediated translational control

Cytoplasmic polyadenylation plays a key role in the translational control of mRNAs driving biological processes such as gametogenesis, cell-cycle progression, and synaptic plasticity. What determines the distinct time of polyadenylation and extent of translational control of a given mRNA, however, is poorly understood. The polyadenylation-regulated translation is controlled by the cytoplasmic polyadenylation element (CPE) and its binding protein, CPEB, which can assemble both translational repression or activation complexes. Using a combination of mutagenesis and experimental validation of genome-wide computational predictions, we show that the number and relative position of two elements, the CPE and the Pumilio-binding element, with respect to the polyadenylation signal define a combinatorial code that determines whether an mRNA will be translationally repressed by CPEB, as well as the extent and time of cytoplasmic polyadenylation-dependent translational activation.     

Early and late functions in a bipartite RNA virus: evidence for translational control by competition between viral mRNAs.

It has been shown previously that Drosophila cells infected with black beetle virus synthesize an early viral protein, protein A, a putative element of the viral RNA polymerase. Synthesis of protein A declines sharply by 6 h postinfection, whereas synthesis of viral coat protein alpha continues for at least 14 h. The early shutoff in protein A synthesis occurred despite the presence of equimolar proportions of the mRNAs for proteins A and alpha, RNAs 1 and 2, respectively. We have now been able to mimic this translational discrimination in a cell-free protein-synthesizing system prepared from infected or uninfected Drosophila cells, thus allowing further analysis of the mechanism by which translation of RNA 1 is selectively turned off. The results revealed no evidence for control by virus-encoded proteins or by virus-induced modification of mRNAs by the cell-free system. Rather, with increasing RNA concentration, viral RNA 1 was outcompeted by its genomic partner, RNA 2. This suggests that the early shutoff in intracellular synthesis of protein A is due to decreasing ability of RNA 1 to compete for a rate-controlling translational factor(s) as the concentration of viral RNAs accumulates within the infected cell.