Solution structure of the Rous sarcoma virus nucleocapsid protein: muPsi RNA packaging signal complex

The 5'-untranslated region (5'-UTR) of retroviral genomes contains elements required for genome packaging during virus assembly. For many retroviruses, the packaging elements reside in non-contiguous segments that span most or all of the 5'-UTR. The Rous sarcoma virus (RSV) is an exception, in that its genome can be packaged efficiently by a relatively short, 82 nt segment of the 5'-UTR called muPsi. The RSV 5'-UTR also contains three translational start codons (AUG-1, AUG-2 and AUG-3) that have been controvertibly implicated in translation initiation and genome packaging, one of which (AUG-3) resides within the muPsi sequence. We demonstrated recently that muPsi is capable of binding to the cognate RSV nucleocapsid protein (NC) with high affinity (dissociation constant K(d) approximately 2 nM), and that residues of AUG-3 are essential for tight binding. We now report the solution structure of the NC:muPsi complex, determined using NMR data obtained for samples containing ((13)C,(15)N)-labeled NC and (2)H-enriched, nucleotide-specifically protonated RNAs. Upon NC binding, muPsi adopts a stable secondary structure that consists of three stem loops (SL-A, SL-B and SL-C) and an 8 bp stem (O3). Binding is mediated by the two zinc knuckle domains of NC. The N-terminal knuckle interacts with a conserved U(217)GCG tetraloop (a member of the UNCG family; N=A,U,G or C), and the C-terminal zinc knuckle binds to residues that flank SL-A, including residues of AUG-3. Mutations of critical nucleotides in these sequences compromise or abolish viral infectivity. Our studies reveal novel structural features important for NC:RNA binding, and support the hypothesis that AUG-3 is conserved for genome packaging rather than translational control.     

Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases

The salvage of deoxyribonucleosides in the social amoeba Dictyostelium discoideum, which has an extremely A+T-rich genome, was investigated. All native deoxyribonucleosides were phosphorylated by D. discoideum cell extracts and we subcloned three deoxyribonucleoside kinase (dNK) encoding genes. D. discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a K(m) of 5.1 microM. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a K(m) for deoxyadenosine of 22.7 microM and a k(cat) of 3.7 s(-1) and could not efficiently phosphorylate any other native deoxyribonucleoside. D. discoideum deoxyguanosine kinase was also a purine-specific kinase and phosphorylated significantly only deoxyguanosine, with a K(m) of 1.4 microM and a k(cat) of 3 s(-1). The two purine-specific deoxyribonucleoside kinases could represent ancient enzymes present in the common ancestor of bacteria and eukaryotes but remaining only in a few eukaryote lineages. The narrow substrate specificity of the D. discoideum dNKs reflects the biased genome composition and we attempted to explain the strict preference of DddAK for deoxyadenosine by modeling the active center with different substrates. Apart from its native substrate, deoxyadenosine, DddAK efficiently phosphorylated fludarabine. Hence, DddAK could be used in the enzymatic production of fludarabine monophosphate, a drug used in the treatment of chronic lymphocytic leukemia.     

Constraining protein sequence space: four amino acid alphabets are sufficient to recapitulate lambda repressor multimerization

Nucleic acid polymers selected from random sequence space constitute an enormous array of catalytic, diagnostic and therapeutic molecules. Despite the fact that proteins are robust polymers with far greater chemical and physical diversity, success in unlocking protein sequence space remains elusive. We have devised a combinatorial strategy for accessing nucleic acid sequence space corresponding to proteins comprising selected amino acid alphabets. Using the SynthOMIC approach (synthesis of ORFs by multimerizing in-frame codons), representative libraries comprising four amino acid alphabets were fused in-frame to the lambda repressor DNA-binding domain to provide an in vivo selection for self-interacting proteins that re-constitute lambda repressor function. The frequency of self-interactors as a function of amino acid composition ranged over five orders of magnitude, from ∼6% of clones in a library comprising the amino acid residues LARE to ∼0.6 in 10⁶ in the MASH library. Sequence motifs were evident by inspection in many cases, and individual clones from each library presented substantial sequence identity with translated proteins by BLAST analysis. We posit that the SynthOMIC approach represents a powerful strategy for creating combinatorial libraries of open reading frames that distils protein sequence space on the basis of three inherent properties: it supports the use of selected amino acid alphabets, eliminates redundant sequences and locally constrains amino acids.     

Influence of the transposable element neighborhood on human gene expression in normal and tumor tissues

Transposable elements (TEs) are genomic sequences able to replicate themselves, and to move from one chromosomal position to another within the genome. Many TEs contain their own regulatory regions, which means that they may influence the expression of neighboring genes. TEs may also be activated and transcribed in various cancers. We therefore tested whether gene expression in normal and tumor tissues is influenced by the neighboring TEs. To do this, we associated all human genes to the nearest TEs. We analyzed the expression of these genes in normal and tumor tissues using SAGE and EST data, and related this to the presence and type of TEs in their vicinity. We confirmed that TEs tend to be located in antisense orientation relative to their hosting genes. We found that the average number of tissues where a gene is expressed varies depending on the type of TEs located near the gene, and that the difference in expression level between normal and tumor tissues is greatest for genes that host SINE elements. This deregulation increases with the number of SINE copies in the gene vicinity. This suggests that SINE elements might contribute to the cascade of gene deregulation in cancer cells.     

Characterization of the first angiotensin-converting like enzyme in bacteria: Ancestor ACE is already active

Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.