Jan van Duin

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.

Scanning model for translational reinitiation in eubacteria

Premature termination of translation in eubacteria, like Escherichia coli, often leads to reinitiation at nearby start codons. Restarts also occur in response to termination at the end of natural coding regions, where they serve to enforce translational coupling between adjacent cistrons. Here, we present a model in which the terminated but not released ribosome reaches neighboring initiation codons by lateral diffusion along the mRNA. The model is based on the finding that introduction of an additional start codon between the termination and the reinitiation site consistently obstructs ribosomes to reach the authentic restart site. Instead, the ribosome now begins protein synthesis at this newly introduced AUG codon. This ribosomal scanning-like movement is bidirectional, has a radius of action of more than 40 nucleotides in the model system used, and activates the first encountered restart site. The ribosomal reach in the upstream direction is less than in the downstream one, probably due to dislodging by elongating ribosomes. The proposed model has parallels with the scanning mechanism postulated for eukaryotic translational initiation and reinitiation.

Single-Stranded RNA Bacteriophages

Since their discovery in 1961 by Loeb and Zinder, the RNA phages have served as a model system to explore a variety of problems in molecular biology. As a source of homogeneous and readily obtainable messenger RNA, they have been particularly helpful in solving questions on initiation of translation, and they have provided good insight into regulation of gene expression at the level of translation. The concepts of translational polarity and translational control by repressor proteins resulted from early studies on bacteriophage RNA.

Visualization by Cryo-electron Microscopy of Genomic RNA that Binds to the Protein Capsid Inside Bacteriophage MS2

The icosahedrally symmetrized structure of bacteriophage MS2 as determined by cryo-electron microscopy (EM) reveals the presence of genomic RNA that attaches to coat-protein dimers. Earlier X-ray diffraction studies revealed similar interactions between the unique operator hairpin of the MS2 genomic RNA and the coat-protein dimer. This observation leads us to conclude that not only the operator, but also many other RNA sequences in the genome of MS2, are able to bind to the coat-protein dimer. A substantial number of potential coat-protein-dimer binding sites are present in the genome of MS2 that can account for the observed RNA densities in the EM map. Moreover, it appears that these stem-loop structures are able to bind in a similar fashion to the coat protein dimer as the wild-type operator hairpin. The EM map also shows additional density between the potential operator-binding sites, linking the RNA stem-loops together to form an icosahedral network around the 3 and 5-fold axes. This RNA network is bound to the inside of the MS2 capsid and probably influences both capsid stability and formation, supporting the idea that capsid formation and RNA packaging are intimately linked to each other.

Determination of the RNA secondary structure that regulates lysis gene expression in bacteriophage MS2

The lysis gene of the RNA bacteriophage MS2 is not expressed unless translation of the overlapping coat gene takes place. To understand the molecular basis for this translational coupling the RNA secondary structure around the lysis gene start was analyzed with structure-specific enzymes and chemicals. The existence of a hairpin between nucleotides 1636 and 1707 is in agreement with the structural mapping data and also with the conservation of base-pairing in the related M12 phage. In this hairpin, the G residues in the Shine and Dalgarno region and start codon are inaccessible to RNase T1, which is consistent with the fact that ribosomal access to the lysis gene is blocked when there is no coat gene translation. Deletions or point mutations that are predicted to destabilize the hairpin give rise to lysis protein synthesis that is independent of coat gene translation. Base substitutions that are not expected to weaken the helix do not lead to independent lysis gene expression. Finally, nucleotide changes that strengthen the hairpin lead neither to uncoupled nor to coupled synthesis of the lysis protein. Structural analysis of mutant MS2 RNA shows that small changes in the stability of the secondary structure lead to substantial differences in translation initiation. The function of the hairpin structure in coupling lysis gene to coat gene translation requires that its stability is kept within narrow limits.

Translational control by delayed RNA folding: Identification of the kinetic trap

The maturation or A-protein gene of single-stranded RNA phage MS2 is preceded by a 130-nt long untranslated leader. When MS2 RNA folding is at equilibrium, the gene is untranslatable because the leader adopts a well-defined cloverleaf structure in which the Shine-Dalgarno (SD) sequence of the maturation gene is taken up in long-distance base pairing with an upstream complementary sequence (UCS). Synthesis of the A-protein takes place transiently while the RNA is synthesized from the minus strand. This requires that formation of the inhibitory cloverleaf is slow. In vitro, the folding delay was on the order of minutes. Here, we present evidence that this postponed folding is caused by the formation of a metastable intermediate. This intermediate is a small local hairpin that contains the UCS in its loop, thereby preventing or slowing down its pairing with the SD sequence. Mutants in which the small hairpin could not be formed made no detectable amounts of A-protein and were barely viable. Apparently, here the cloverleaf formed quicker than ribosomes could bind. On the other hand, mutants in which the small intermediary hairpin was stabilized produced more A-protein than wild type and were viable. One hardly growing mutant that could not form the metastable hairpin and did not make detectable amounts of A-protein was evolved. The emerging pseudo-revertant had selected two second site repressor mutations that allowed reconstruction of a variant of the metastable intermediate. The pseudo-revertant had also regained the capacity to produce the A-protein.

Phage display selects for amylases with improved low pH starch-binding

Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type alpha-amylase. These data demonstrate that useful alpha-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values.

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.

Translational regulation of the lysis gene in RNA bacteriophage fr requires a UUG initiation codon

SummarySingle nucleotide substitutions identify a UUG triplet as the initiation codon of the lysis gene in RNA bacteriophage fr. This initiation codon is non-functional in de novo initiation but is activated by translational termination at the overlapping coat gene. The UUG initiation codon is crucial for gene regulation in the phage, as it excludes uncontrolled access of ribosomes to the start of the lysis gene. Replacement of UUG by either GUG or AUG results in the loss of genetic control of the lysis gene. A model is presented in which initiation factor IF3 proofreads de novo initiation at UUG codons.