Maarten H. de Smit

Control of Prokaryotic Translational Initiation by mRNA Secondary Structure

Publisher Summary This chapter describes the evidence that differences in the secondary structures of RNA are probably the main cause of this unpredictability. Although it has been known for many years that secondary structures of mRNA can interfere with translational initiation, it is quite surprising to find a simple, linear relationship between the efficiency of a ribosome binding site and the fraction of unfolded mRNA molecules. Presumably, a non-sequence-specific interaction of ribosomes with single-stranded RNA constitutes the first step in the process of initiation. The existence of such an interaction is supported by several lines of evidence. For example, the binding of ribosomes to synthetic polynucleotides like poly(U) must rely solely on nonspecific contacts, yet results in efficient polypeptide synthesis. Several sophisticated mechanisms of translational regulation that function through reversible changes in inhibitory secondary structures have been elucidated and are discussed in this chapter.

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 16 S 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.

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 tRNAAla 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 G13 and U342. 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.

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.

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 lacZ 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.

Control of Translational Initiation by mRNA Secondary Structure: A Quantitative Analysis

There is good evidence that mRNA secondary structure is one of the main factors determining the efficiency of translations initiation in prokaryotes (reviewed by Stormo, 1986; Gold, 1988; de Smit & van Duin, 1990). For example, Hall et al. (1982) found that mutations stabilizing a potential hairpin structure in the ribosome binding site of the lamB gene inhibited its expression and this inhibition could be relieved by second-site destabilizing mutations. They further suggested that the level of expression in the different mutants was related to the relative stability of the helix. Similarly, others showed the expression of heterologous genes in E. coli to be related to the stability of defined secondary structures involving the ribosome binding sites (Buell et al., 1985; Tessier et al., 1984; Spanjaard et al., 1989).