Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1-the largest protein in the complex-as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1 - the largest protein in the complex - as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

Update on Distribution and Genetic Variability of Plum pox virus Strains in Bulgaria.

Field surveys for Plum pox virus (PPV) infection were conducted in stone fruit orchards all over Bulgaria. In total, 1168 out of 3020 leaf samples from cultivated Prunus spp. and wildly growing P. cerasifera trees reacted positive for PPV in DASI-ELISA with the universal monoclonal antibody (MAb) 5B. Further ELISA analyses showed that 987 and 127 isolates belonged to PPV-M and PPV-D serotypes, respectively. The plum and P. cerasifera showed 82.0% and 50.5% levels of infection, respectively followed by the peach (40.0%) and the apricot (32.0%). Five hundred fifty one PPV isolates were further typed by IC-RT-PCR with PPV-Rec, -M and -D-specific primers, targeting (Cter)NIb-(Nter) CP genome region, as 125 isolates were sequenced. The results revealed the presence of PPV-Rec, PPV-M and PPV-D and mixed infections of these strains. PPV-Rec was the most prevalent strain (49.0%), followed by PPV-M (40.1%), while PPV-D was the less spread strain (8.2%). PPV-Rec was the most common strain in plums, including the eight "old-aged" trees from the region of the first Sharka discovery. PPV-M was the most prevalent strain in peach and apricot. Phylogenetic analyses on (Cter)NIb-(Nter)CP of the isolates were performed. PPV-Rec isolates formed a homogeneous group, while PPV-M isolates split into PPV-Ma and PPV-Mb subgroups. Five separated clades were formed by the analyzed PPV-D isolates. Nucleotide sequences of the partial CP coding region of the analyzed isolates revealed a slightly higher intra-strain genetic variability in PPV-Rec and PPV-M isolates, while that of PPV-D strain isolates was higher from the reported for these strains.

Co-translational assembly of mammalian nuclear multisubunit complexes.

Cells dedicate significant energy to build proteins often organized in multiprotein assemblies with tightly regulated stoichiometries. As genes encoding subunits assembling in a multisubunit complex are dispersed in the genome of eukaryotes, it is unclear how these protein complexes assemble. Here, we show that mammalian nuclear transcription complexes (TFIID, TREX-2 and SAGA) composed of a large number of subunits, but lacking precise architectural details are built co-translationally. We demonstrate that dimerization domains and their positions in the interacting subunits determine the co-translational assembly pathway (simultaneous or sequential). The lack of co-translational interaction can lead to degradation of the partner protein. Thus, protein synthesis and complex assembly are linked in building mammalian multisubunit complexes, suggesting that co-translational assembly is a general principle in mammalian cells to avoid non-specific interactions and protein aggregation. These findings will also advance structural biology by defining endogenous co-translational building blocks in the architecture of multisubunit complexes.

Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription.

Mediterranean and central-eastern European countries host viruses of two different clades of plum pox virus strain M.

The genetic diversity of plum pox virus strain M (PPV-M) was assessed by analyzing 28 isolates collected in 8 European countries. Two genomic fragments spanning the (Cter)P3-6K1-(Nter)CI coding region as well as the full coat protein coding region were sequenced directly from PCR products. Phylogenetic analysis showed that the geographical origin of the collected isolates was clearly associated with two different PPV-M clades. Moreover, the pattern of substitutions in the CP gene shed light on the evolutionary relationships between PPV-M and the recombinant strains PPV-Rec and PPV-T.

Identification and Characterization of a Novel Tobamovirus from Tropical Soda Apple in Florida.

Foliar symptoms suggestive of virus infection were recently observed on the noxious weed tropical soda apple (Solanum viarum) in Florida. An agent was mechanically transmitted to Nicotiana benthamiana, and virions were isolated from systemically infected leaves. Rod-shaped particles ~300 nm in length were observed in the partially purified preparations by electron microscopy. The host range determined by mechanical inoculation with purified virions included all tested plants in the Solanaceae (16 species including the important vegetable crops, pepper and tomato) and Chenopodiaceae (2 species) but excluded all tested plants in the Ama-ranthaceae, Apocynaceae, Brassicaceae, Caryophyllaceae, Cucurbitaceae, Fabaceae, Lamiaceae, Malvaceae, and Tropaeolaceae, including several common virus indicator hosts. Comparisons of the coat and movement protein nucleotide and deduced amino acid sequences of this putative tobamovirus with recognized members of this genus, indicate that it is a novel tobamovirus that shares the highest level of sequence identity with Pepper mild mottle virus followed by other members of the Solanaceae-infecting subgroup of tobamoviruses. The virus, for which the name Tropical soda apple mosaic virus (TSAMV) is proposed, was found to be widespread in tropical soda apple in peninsular Florida during an initial survey. TSAMV contamination of seed from infected tropical soda apple plants was found, suggesting that seed transmission may be important for TSAMV dissemination and epidemiology.

Comparison of Detection Methods for a Novel Tobamovirus Isolated from Florida Hibiscus.

A novel tobamovirus recently was isolated from hibiscus in Florida. Serological and molecular methods, including enzyme-linked immunosorbent assay (ELISA), dot-blot immunoassay (DBIA), tissue-blot immunoassay (TBIA), and immunocapture reverse-transcription poly-merase chain reaction (IC-RT-PCR) were compared to evaluate their usefulness for diagnosis of this virus. Each method was tested with partially purified virus preparations and tissue samples from infected hibiscus and Chenopodium quinoa plants. Indirect ELISA was more sensitive than double-antibody sandwich (DAS)-ELISA with all samples tested. The Florida hibiscus virus was detectable in hibiscus leaves and bark up to 1:12,800 and 1:6,400 dilutions, respectively, by indirect ELISA and up to 1:3,200 and 1:400 dilutions by DAS-ELISA. End-point dilutions of partially purified virus preparations from indirect and DAS-ELISA were 4 and 31 ng/ml, respectively. Florida hibiscus virus was detected by DBIA in sap from hibiscus bark and leaves at dilutions up to 1:400 and 1:800, respectively, showing that DBIA was less sensitive than either ELISA method. The virus also was detected reliably by TBIA from leaves and bark of hibiscus plants. The most sensitive method was IC-RT-PCR, which could detect as little as 500 pg/ml of virus in partially purified preparations and was 16- and 32-fold more sensitive than DAS-ELISA with hibiscus bark and leaf extracts, respectively. Over 600 hibiscus samples were tested by various combinations of these methods to validate their usefulness.

Transmission, In Planta Distribution, and Management of Hibiscus latent Fort Pierce virus, a Novel Tobamovirus Isolated from Florida Hibiscus.

Three aspects of the infection process of a new tobamovirus species, Hibiscus latent Fort Pierce virus, recently isolated from hibiscus in Florida, were examined: (i) transmission efficiency of rub-, slash-, and cut-inoculation for two hibiscus cultivars, Pink Versicolor and Brilliant Red; (ii) distribution within infected hibiscus plants; and (iii) treatments to prevent infection during plant propagation and pruning. Rub-, slash-, and cut-inoculation methods were all effective and yielded infection rates of 66, 74, and 70%, respectively, in Pink Versicolor and 50, 56, and 38%, respectively, in Brilliant Red. Analysis of virus distribution in infected plants over time revealed that the virus moved from the place of inoculation to the roots and then toward the bottom (oldest) leaves of the plants. Virus was found in all leaves on branches of Brilliant Red plants at 210 days postinoculation, whereas it remained restricted to the bottom and middle leaves of Pink Versicolor plants at 290 days postinoculation. Although several treatments of tools reduced infection of hibiscus during experiments mimicking plant propagation and pruning, 10% (wt/vol) sodium hypochlorite and 20% (wt/vol) nonfat dry milk completely prevented infection.

Biological and Molecular Characterization of a Novel Tobamovirus with a Unique Host Range.

Tobamoviruses are among the best characterized and most studied plant viruses. Three subgroups of tobamoviruses correspond to viral genome sequence and host range to include those viruses infecting (i) solanaceous plants, (ii) brassicas, or (iii) cucurbits or legumes. We isolated a virus from Florida landscape plantings of the malvaceous plant hibiscus (Hibiscus rosasinensis) that appears to be a tobamovirus based upon its virion morphology, genome organization, and coat protein sequence. The experimental host range of this virus included five malvaceous species but excluded all tested brassica, cucurbit, and legume species and 12 of the 19 solanaceous species tested. The unique host range and comparison of coat protein gene and protein sequences with those of recognized tobamoviruses indicate that this is a novel to-bamovirus. A limited survey revealed that this virus is widespread in hibiscus and related species in the Florida landscape.