Apoptosis-inducing proteins in chicken anemia virus and TT virus.

Torque teno viruses (TTVs) share several genomic similarities with the chicken anemia virus (CAV). CAV encodes the protein apoptin that specifically induces apoptosis in (human) tumor cells. Functional studies reveal that apoptin induces apoptosis in a very broad range of (human) tumor cells. A putative TTV open reading frame (ORF) in TTV genotype 1, named TTV apoptosis inducing protein (TAIP), it induces, like apoptin, p53-independent apoptosis in various human hepatocarcinoma cell lines to a similar level as apoptin. In comparison to apoptin, TAIP action is less pronounced in several analyzed human non-hepatocarcinoma-derived cell lines. Detailed sequence analysis has revealed that the TAIP ORF is conserved within a limited group of the heterogeneous TTV population. However, its N-terminal half, N-TAIP, is rather well conserved in a much broader set of TTV isolates. The similarities between apoptin and TAIP, and their relevance for the development and treatment of diseases is discussed.

Structural parameters affecting the kinetics of RNA hairpin formation.

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program 'Kinfold'. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem-loop structures.

Rapid evolution of translational control mechanisms in RNA genomes.

We have introduced 13 base substitutions into the coat protein gene of RNA bacteriophage MS2. The mutations, which are clustered ahead of the overlapping lysis cistron, do not change the amino acid sequence of the coat protein, but they disrupt a local hairpin, which is needed to control translation of the lysis gene. The mutations decreased the phage titer by four orders of magnitude but, upon passaging, the virus accumulated suppressor mutations that raised the fitness to almost wild-type level. Analysis of the pseudorevertants showed that the disruption of the local hairpin, controlling expression of the lysis gene, had apparently been so complete that its restoration by chance mutations could not be achieved. Instead, alternative foldings initiated by the starting mutations were further stabilized and optimized. Strikingly, in the pseudorevertants analyzed, translational control of the lysis gene had been restored. This feat was accomplished by, on average, four suppressor mutations that generally occurred at codon wobble positions. We also introduced 11 mutations in a hairpin more upstream in the coat protein gene and not implicated in lysis control. Here the titer dropped by three logs, but pseudorevertants with a fitness close to wild-type were soon generated. These pseudorevertants again were the result of the optimization of alternative foldings induced by the mutations. The transition of the secondary structure from wild-type to pseudorevertant could be visualized by structure probing. Our study shows that the folding of the RNA is an important phenotypic property of RNA viruses. However, its distortion can easily be overcome by optimizing alternative base-pairings. These new structures are not qualitatively equivalent to the original one, since they do not successfully compete with the wild-type.

Isolation, renaturation and partial characterization of recombinant human transferrin and its half molecules from Escherichia coli.

Recombinant human transferrin as well as N- and C-terminal half-transferrins, produced in Escherichia coli, are deposited in inclusion bodies by the bacteria. The isolation and purification of the recombinant proteins from these inclusion bodies are described here. The amino acid compositions and N-terminal sequences of the proteins were determined, and found to be in agreement with the known protein structure of human serum transferrin. Renaturation of the recombinant proteins is described, resulting in water-soluble iron-binding molecules. Iron binding was confirmed by 59Fe labelling, absorption spectrophotometry and EPR spectrometry.

Optimized bacterial production of nonglycosylated human transferrin and its half-molecules.

Transferrin is a glycoprotein functioning in iron transport in higher eukaryotes, and consists of two highly homologous domains. To study the function of the glycan residues attached exclusively to the C-terminal domain, we have constructed a plasmid allowing production of nonglycosylated human transferrin in Escherichia coli. By molecular biological and genetic techniques, production was stepped up to 60 mg/l. Similar plasmids were constructed for production of the two half-transferrins. The recombinant proteins accumulate in inclusion-body-like aggregates, where they appear to bind iron without causing bacteriostasis. Proteins active in iron binding have been purified from these inclusion bodies.

Control of translation by mRNA secondary structure in Escherichia coli. A quantitative analysis of literature data.

Translational efficiency in Escherichia coli is strongly controlled by the secondary structure of the mRNA in the translational initiation region. We have previously shown that protein production from the coat-protein gene of RNA bacteriophage MS2 is directly related to the fraction of mRNA molecules in which the ribosome binding site is unfolded. This fraction is dictated by the free energy (delta Gf0) of the local secondary structure. We now present a similar analysis of published data on four other ribosome binding sites. The results conform quantitatively to the same relationship as found for the MS2 coat-protein gene. The efficiency of translation is determined by the overall stability of the structure at the ribosome binding site, whether the initiation codon itself is base-paired or not. Structures weaker than -6 kcal/mol usually do not reduce translational efficiency. Below this threshold, all systems show a tenfold decrease in expression for every -1.4 kcal/mol, as predicted from theory.

CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli.

To activate expression of a human transferrin (Tf)-encoding cDNA in Escherichia coli by translational coupling, it was placed in an expression plasmid downstream from a 5'-terminal fragment from the replicase (R)-encoding gene of bacteriophage MS2. The resulting construct was found to produce, besides the desired Tf, a protein with the mobility of a fusion product (RTf) of the N-terminal R fragment and Tf. Analysis of available mutants showed that this fusion results from +1 ribosomal frameshifting at the end of the R reading frame. This region contains the sequence, CCC.UGA, suggesting that before termination occurs, tRNA(Pro) may dislodge from the CCC codon and reassociate with the +1 triplet CCU. By further site-directed mutagenesis, we demonstrate that both the CCC codon and the termination codon are indeed required for the observed 2-4% frameshifting. When either triplet is changed, the frequency of frameshifting drops to 0.3% or less. These results classify CCC.UGA as a new '+1 shifty stop'.

Translational initiation on structured messengers. Another role for the Shine-Dalgarno interaction.

Translational efficiency in Escherichia coli is in part determined by the Shine-Dalgarno (SD) interaction, i.e. the base-pairing of the 3' end of 16S ribosomal RNA to a stretch of complementary nucleotides in the messenger, located just upstream of the initiation codon. Although a large number of mutations in SD sequences have been produced and analysed, it has so far not been possible to find a clear-cut quantitative relationship between the extent of the complementarity to the rRNA and translational efficiency. This is presumably due to a lack of information about the secondary structures of the messengers used, before and after mutagenesis. Such information is crucial, because intrastrand base-pairing of a ribosome binding site can have a profound influence on its translational efficiency. By site-directed mutagenesis, we have varied the extent of the SD complementarity in the coat-protein gene of bacteriophage MS2. The ribosome binding site of this gene is known to adopt a simple hairpin structure. Substitutions in the SD region were combined with other mutations, which altered the stability of the structure in a predictable way. We find that mutations reducing the SD complementarity by one or two nucleotides diminish translational efficiency only if ribosome binding is impaired by the structure of the messenger. In the absence of an inhibitory structure, these mutations have no effect. In other words, a strong SD interaction can compensate for a structured initiation region. This can be understood by considering translational initiation on a structured ribosome binding site as a competition between intramolecular base-pairing of the messenger and binding to a 30 S ribosomal subunit. A good SD complementarity provides the ribosome with an increased affinity for its binding site, and thereby enhances its ability to compete against the secondary structure. This function of the SD interaction closely parallels the RNA-unfolding capacity of ribosomal protein S1. By comparing the expression data from mutant and wild-type SD sequences, we have estimated the relative contribution of the SD base-pairs to ribosome-mRNA affinity. Quantitatively, this contribution corresponds quite well with the theoretical base-pairing stabilities of the wild-type and mutant SD interactions.

Translational initiation at the coat-protein gene of phage MS2: native upstream RNA relieves inhibition by local secondary structure.

Maximal translation of the coat-protein gene from RNA bacteriophage MS2 requires a contiguous stretch of native MS2 RNA that extends hundreds of nucleotides upstream from the translational start site. Deletion of these upstream sequences from MS2 cDNA plasmids results in a 30-fold reduction of translational efficiency. By site-directed mutagenesis, we show that this low level of expression is caused by a hairpin structure centred around the initiation codon. When this hairpin is destabilized by the introduction of mismatches, expression from the truncated messenger increases 20-fold to almost the level of the full-length construct. Thus, the translational effect of hundreds of upstream nucleotides can be mimicked by a single substitution that destabilizes the structure. The same hairpin is also present in full-length MS2 RNA, but there it does not impair ribosome binding. Apparently, the upstream RNA somehow reduces the inhibitory effect of the structure on translational initiation. The upstream MS2 sequence does not stimulate translation when cloned in front of another gene, nor can unrelated RNA segments activate the coat-protein gene. Several possible mechanisms for the activation are discussed and a function in gene regulation of the phage is suggested.

Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis.

We have quantitatively analyzed the relationship between translational efficiency and the mRNA secondary structure in the initiation region. The stability of a defined hairpin structure containing a ribosome binding site was varied over 12 kcal/mol (1 cal = 4.184 J) by site-directed mutagenesis and the effects on protein yields were analyzed in vivo. The results reveal a strict correlation between translational efficiency and the stability of the helix. An increase in its delta G0 of -1.4 kcal/mol (i.e., less than the difference between an A.U and a G.C pair) corresponds to the reduction by a factor of 10 in initiation rate. Accordingly, a single nucleotide substitution led to the decrease by a factor of 500 in expression because it turned a mismatch in the helix into a match. We find no evidence that exposure of only the Shine-Dalgarno region or the start codon preferentially favors recognition. Translational efficiency is strictly correlated with the fraction of mRNA molecules in which the ribosome binding site is unfolded, indicating that initiation is completely dependent on spontaneous unfolding of the entire initiation region. Ribosomes appear not to recognize nucleotides outside the Shine-Dalgarno region and the initiation codon.

Secondary structure of the central region of bacteriophage MS2 RNA. Conservation and biological significance.

The RNA of the Escherichia coli RNA phages is highly structured with 75% of the nucleotides estimated to take part in base-pairing. We have used enzymatic and chemical sensitivity of nucleotides, phylogenetic sequence comparison and the phenotypes of constructed mutants to develop a secondary structure model for the central region (900 nucleotides) of the group I phage MS2. The RNA folds into a number of, mostly irregular, helices and is further condensed by several long-distance interactions. There is substantial conservation of helices between the related groups I and II, attesting to the relevance of discrete RNA folding. In general, the secondary structure is thought to be needed to prevent annealing of plus and minus strand and to confer protection against RNase. Superimposed, however, are features required to regulate translation and replication. The MS2 RNA section studied here contains three translational start sites, as well as the binding sites for the coat protein and the replicase enzyme. Considering the density of helices along the RNA, it is not unexpected to find that all these sites lie in helical regions. This fact, however, does not mean that these sites are recognized as secondary structure elements by their interaction partners. This holds true only for the coat protein binding site. The other four sites function in the unfolded state and the stability of the helix in which they are contained serves to negatively control their accessibility. Mutations that stabilize helices containing ribosomal binding sites reduce their efficiency and vice versa. Comparison of homologous helices in different phage RNAs indicates that base substitutions have occurred in such a way that the thermodynamic stability of the helix is maintained. The evolution of individual helices shows several distinct size-reduction patterns. We have observed codon deletions from loop areas and shortening of hairpins by base-pair deletions from either the bottom, the middle or the top of stem structures. Evidence for the coaxial stacking of some helical segments is discussed.

The amino terminal half of the MS2-coded lysis protein is dispensable for function: implications for our understanding of coding region overlaps.

We have asked whether genetic overlaps only evolve to provide extra coding capacity in genomes of restricted size. As a model system we have used the lysis gene of the RNA bacteriophage MS2. This gene overlaps with the distal part of the coat protein gene and with the proximal part of the replicase gene. Using recombinant DNA procedures we have determined whether either of the two overlaps codes for amino acids that are not essential for the function of the 75 amino acid long lysis protein. We find that the first 40 amino acids of the lysis protein are dispensable for function. Thus all of the genetic information essential to the synthesis of the active C-terminal peptide lies within the overlap with the replicase gene, whereas all dispensable residues are encoded in the overlap with the coat protein gene and in the intercistronic region. This suggests that the overlap with the coat protein gene is not required for extra coding capacity but serves to regulate the expression of the lysis gene. Comparative sequence analysis is consistent with this idea.