In vivo dynamics of intracistronic transcriptional polarity.

Transcriptional polarity occurs in Escherichia coli when cryptic Rho-dependent transcription terminators become activated as a consequence of reduced translation. Increased spacing between RNA polymerase and the leading ribosome allows the transcription termination factor Rho to bind to mRNA, migrate to the RNA polymerase, and induce termination. Transcriptional polarity results in decreased synthesis of inefficiently translated mRNAs and, therefore, in decreased expression not only of downstream genes in the same operon (intercistronic polarity) but also of the cistron in which termination occurs (intracistronic polarity). To quantitatively measure the effect of different levels of translation on intracistronic transcription termination, the polarity-prone lacZ reporter gene was fused to a range of mutated ribosome binding sites, repressed to different degrees by local RNA structure. The results show that polarity gradually increases with decreasing frequency of translational initiation, as expected. Closer analysis, with the help of a newly developed kinetic model, reveals that efficient intracistronic termination requires very low translational initiation frequencies. This finding is unexpected because Rho is a relatively small protein that binds rapidly to its RNA target, but it appears to be true also for other examples of transcriptional polarity reported in the literature. The conclusion must be that polarity is more complex than just an increased exposure of the Rho binding site as the spacing between the polymerase and the leading ribosome becomes larger. Biological consequences and possible mechanisms are discussed.

Intracistronic transcriptional polarity enhances translational repression: a new role for Rho.

Transcriptional polarity in Escherichia coli occurs when cryptic Rho-dependent transcription terminators become activated as a consequence of reduced translation. Whether this is due to an increased spacing between the RNA polymerase and the leading ribosome or to prior functional inactivation of a subpopulation of the mRNAs has been a matter of discussion. Transcriptional polarity results in decreased synthesis of inefficiently translated mRNAs and therefore in decreased expression of downstream genes in the same operon (intercistronic polarity). By analogy, expression of the gene in which the conditional termination occurs is also expected to decrease, but this has so far not been demonstrated experimentally. To study the relevance of this intracistronic polarity for expression regulation in vivo, the polarity-prone IacZ reporter gene was fused to a range of mutated ribosome binding sites, repressed to different degrees by local RNA structure. Quantitative analysis of protein and mRNA synthesis shows that polarity occurs on functionally active mRNA molecules and that it indeed affects expression of the cistron carrying the terminator, thus enhancing the effect of translational repression. These findings point to a novel regulatory function of transcriptional polarity, reminiscent of transcriptional attenuation but opposite in effect.

Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients.

Multiplex ligation-dependent probe amplification (MLPA) is widely used to screen genes of interest for deletions and duplications. Since MLPA is usually based on size-separation of the amplification products, the maximum number of target sequences that can be screened in parallel is usually limited to approximately 40. We report the design of a robust array-based MLPA format that uses amplification products of essentially uniform size (100-120 bp) and distinguishes between them by virtue of incorporated tag sequences. We were thus able to increase probe complexity to 124, with very uniform product yields and signals that have a low coefficient of variance. The assay designed was used to screen the largest set studied so far (249 patients) of unrelated Duchenne muscular dystrophy (DMD) cases from the Chinese population. In a blind study we correctly assigned 98% of the genotypes and detected rearrangements in 181 cases (73%); i.e., 163 deletions (65%), 13 duplications (5%), and five complex rearrangements (2%). Although this value is significantly higher for Chinese patients than previously reported, it is similar to that found for other populations. The location of the rearrangements (76% in the major deletion hotspot) is also in agreement with other findings. The 96-well flow-through microarray system used in this research provides high-throughput and speed; hybridization can be completed in 5 to 30 minutes. Since array processing and data analysis are fully automated, array-MLPA should be easy to implement in a standard diagnostic laboratory. The universal array can be used to analyze any tag-modified MLPA probe set.

Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles.

A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.

Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA.

We have previously shown that stable base-pairing at a translational initiation site in Escherichia coli can inhibit translation by competing with the binding of ribosomes. When the base-pairing is not too strong, this competition is won by the ribosomes, resulting in efficient translation from a structured ribosome binding site (RBS). We now re-examine these results in the light of RNA folding kinetics and find that the window during which a folded RBS is open is generally much too short to recruit a 30S ribosomal subunit from the cytoplasm. We argue that to achieve efficient expression, a 30S subunit must already be in contact with the mRNA while this is still folded, to shift into place as soon as the structure opens. Single-stranded regions flanking the structure may constitute a standby site, to which the 30S subunit can attach non-specifically. We propose a steady-state kinetic model for the early steps of translational initiation and use this to examine various quantitative aspects of standby binding. The kinetic model provides an explanation of why the earlier equilibrium competition model predicted implausibly high 30S-mRNA affinities. Because all RNA is structured to some degree, standby binding is probably a general feature of translational initiation.

The effect of lamivudine on the replication of hepatitis B virus in HIV-infected patients depends on the host immune status (CD4 cell count).

We retrospectively analysed the effect of lamivudine on hepatitis B virus (HBV) in HIV-co-infected patients. In this small study we document a CD4 cell-dependent response on HBV replication with lamivudine therapy. In this respect, hepatitis B may be considered an opportunistic infection. Our finding may have important clinical consequences, and large prospective studies are necessary to investigate this further.

Structural variation and functional importance of a D-loop-T-loop interaction in valine-accepting tRNA-like structures of plant viral RNAs.

Valine-accepting tRNA-like structures (TLSs) are found at the 3' ends of the genomic RNAs of most plant viruses belonging to the genera Tymovirus, Furovirus, Pomovirus and Pecluvirus, and of one Tobamovirus species. Sequence alignment of these TLSs suggests the existence of a tertiary D-loop-T-loop interaction consisting of 2 bp, analogous to those in the elbow region of canonical tRNAs. The conserved G(18).Psi(55) pair of regular tRNAs is found to covary in these TLSs between G.U (possibly also modified to G.Psi) and A.G. We have mutated the relevant bases in turnip yellow mosaic virus (TYMV) and examined the mutants for symptom development on Chinese cabbage plants and for accumulation of genetic reversions. Development of symptoms is shown to rely on the presence of either A.G or G.U in the original mutants or in revertants. This finding supports the existence and functional importance of this tertiary interaction. The fact that only G.U and A.G are accepted at this position appears to result from steric and energetic limitations related to the highly compact nature of the elbow region. We discuss the implications of these findings for the various possible functions of the valine-accepting TLS.

Functional evidence for D- and T-loop interactions in tmRNA.

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNA(Ala) and also has a clover-leaf structure folded similarly as in canonical tRNAs. Here we propose the L-shape of tmRNA to be stabilized by two tertiary interactions between its D- and T-loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G(13) and U(342). Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.