Structural features of the tmRNA-ribosome interaction.

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

Structural aspects of trans-translation.

trans-Translation is a process which the bacterial cells apply to rescue the ribosomes that are arrested during the translation of damaged mRNA and to get rid of the mRNA and the product polypeptide. In the course of trans-translation, the mRNA-like domain of tmRNA replaces the nonstop messenger RNA bound to the ribosome. Although several structural elements of tmRNA and SmpB known to be essential for correct determination of resume codon, the molecular mechanism of trans-translation is not well understood. Computer modeling has been used to develop a model for the spatial organization of the tmRNA inside the ribosome at different stages of trans-translation leading to a proposal for the mechanism of the template-switching process.

One SmpB molecule accompanies tmRNA during its passage through the ribosomes.

tmRNA and SmpB are the main participants of trans-translation, a process which rescues the ribosome blocked during translation of non-stop mRNA. While a one-to-one stoichiometry of tmRNA to the ribosome is generally accepted, the number of SmpB molecules in the complex is still under question. We have isolated tmRNA-ribosome complexes blocked at different steps of the tmRNA path through the ribosome and analyzed the stoichiometry of the complexes. Ribosome, tmRNA and SmpB were found in equimolar amount in the tmRNA-ribosome complexes stopped at the position of the 2nd, 4th, 5th or the 11th codons of the coding part of the tmRNA.

Comparison of characteristics and function of translation termination signals between and within prokaryotic and eukaryotic organisms.

Six diverse prokaryotic and five eukaryotic genomes were compared to deduce whether the protein synthesis termination signal has common determinants within and across both kingdoms. Four of the six prokaryotic and all of the eukaryotic genomes investigated demonstrated a similar pattern of nucleotide bias both 5' and 3' of the stop codon. A preferred core signal of 4 nt was evident, encompassing the stop codon and the following nucleotide. Codons decoded by hyper-modified tRNAs were over-represented in the region 5' to the stop codon in genes from both kingdoms. The origin of the 3' bias was more variable particularly among the prokaryotic organisms. In both kingdoms, genes with the highest expression index exhibited a strong bias but genes with the lowest expression showed none. Absence of bias in parasitic prokaryotes may reflect an absence of pressure to evolve more efficient translation. Experiments were undertaken to determine if a correlation existed between bias in signal abundance and termination efficiency. In Escherichia coli signal abundance correlated with termination efficiency for UAA and UGA stop codons, but not in mammalian cells. Termination signals that were highly inefficient could be made more efficient by increasing the concentration of the cognate decoding release factor.

Transient erythromycin resistance phenotype associated with peptidyl-tRNA drop-off on early UGG and GGG codons.

Expression of minigenes encoding tetra- or pentapeptides MXLX or MXLXV (E peptides), where X is a nonpolar amino acid, renders cells erythromycin resistant whereas expression of minigenes encoding tripeptide MXL does not. By using a 3A' reporter gene system beginning with an E-peptide-encoding sequence, we asked whether the codons UGG and GGG, which are known to promote peptidyl-tRNA drop-off at early positions in mRNA, would result in a phenotype of erythromycin resistance if located after this sequence. We find that UGG or GGG, at either position +4 or +5, without a following stop codon, is associated with an erythromycin resistance phenotype upon gene induction. Our results suggest that, while a stop codon at +4 gives a tripeptide product (MIL) and erythromycin sensitivity, UGG or GGG codons at the same position give a tetrapeptide product (MILW or MILG) and phenotype of erythromycin resistance. Thus, the drop-off event on GGG or UGG codons occurs after incorporation of the corresponding amino acid into the growing peptide chain. Drop-off gives rise to a peptidyl-tRNA where the peptide moiety functionally mimics a minigene peptide product of the type previously associated with erythromycin resistance. Several genes in Escherichia coli fulfill the requirements of high mRNA expression and an E-peptide sequence followed by UGG or GGG at position +4 or +5 and should potentially be able to give an erythromycin resistance phenotype.

Functional interplay between the jaw domain of bacterial RNA polymerase and allele-specific residues in the product RNA-binding pocket.

Bacterial RNA polymerase (RNAP) is a complex molecular machine in which the network of interacting parts and their movements, including contacts to nascent RNA and the DNA template, are at best partially understood. The jaw domain is a part of RNAP that makes a key contact to duplex DNA as it enters the enzyme from downstream and also contacts two other parts of RNAP, the trigger loop, which lies in the RNAP secondary channel, and a sequence insertion in the Escherichia coli RNAP trigger loop that forms an external domain and also contacts downstream DNA. Deletion of the jaw domain causes defects in transcriptional pausing and in bacterial growth. We report here that these defects can be partially corrected by a limited set of substitutions in a distant part of RNAP, the product RNA-binding pocket. The product RNA-binding pocket binds nascent RNA upstream of the active site and is the binding site for the RNAP inhibitor rifampicin when RNA is absent. These substitutions have little effect on transcript elongation between pause sites and actually exacerbate jaw-deletion defects in transcription initiation, suggesting that the pausing defects may be principally responsible for the in vivo phenotype of the jaw deletion. We suggest that the counteracting effects on pausing of the alterations in the jaw and the product RNA binding site may be mediated either by effects on translocation or via allosteric communication to the RNAP active site.

RNA chaperone activity of translation initiation factor IF1.

Translation initiation factor IF1 is an indispensable protein for translation in prokaryotes. No clear function has been assigned to this factor so far. In this study we demonstrate an RNA chaperone activity of this protein both in vivo and in vitro. The chaperone assays are based on in vivo or in vitro splicing of the group I intron in the thymidylate synthase gene (td) from phage T4 and an in vitro RNA annealing assay. IF1 wild-type and mutant variants with single amino acid substitutions have been analyzed for RNA chaperone activity. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Furthermore, both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.

A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli.

The influences on gene expression by codons at positions +2, +3, +5 and +7 downstream of the initiation codon have been compared. Most of the +2 codons that are known to give low gene expression are associated with a higher expression if placed at the later positions. The NGG codons AGG, CGG, UGG and GGG, but not GGN or GNG (where N is non-G), are unique since they are associated with a very low gene expression also if located at positions +2, +3 and +5. All codons, including NGG, give a normal gene expression if placed at positions +7. The negative effect by the NGG codons is true for both the lacZ and 3A' model genes. The low expression is suggested to originate at the translational level, although it is not the result of mRNA secondary structure or a lowered intracellular mRNA pool.

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level.

The influence in vivo of mutated forms of translation initiation factor (IF1) on the expression of the lacZ or 3A' reporter genes, with different initiation and/or +2 codons, has been investigated. Reporter gene expression in these infA(IF1) mutants is similar to the wild-type strain. The results do not support the longstanding hypothesis that IF1 could perform discriminatory functions while blocking the aminoacyl-tRNA acceptor site (A-site) of the ribosome. One cold-sensitive IF1 mutant shows a general overexpression, in particular at low temperatures, of both reporter genes at the protein but not mRNA level.

Abortive translation caused by peptidyl-tRNA drop-off at NGG codons in the early coding region of mRNA.

In Escherichia coli the codons CGG, AGG, UGG or GGG (NGG codons) but not GGN or GNG (where N is non-G) are associated with low expression of a reporter gene, if located at positions +2 to +5. Induction of a lacZ reporter gene with any one of the NGG codons at position +2 to +5 does not influence growth of a normal strain, but growth of a strain with a defective peptidyl-tRNA hydrolase (Pth) enzyme is inhibited. The same codons, if placed at position +7, did not give this effect. Other codons, such as CGU and AGA, at location +2 to +5, did not give any growth inhibition of either the wild-type or the mutant strain. The inhibitory effect on the pth mutant strain by NGG codons at location +5 was suppressed by overexpression of the Pth enzyme from a plasmid. However, the overexpression of cognate tRNAs for AGG or GGG did not rescue from the growth inhibition associated with these codons early in the induced model gene. The data suggest that the NGG codons trigger peptidyl-tRNA drop-off if located at early coding positions in mRNA, thereby strongly reducing gene expression. This does not happen if these codons are located further down in the mRNA at position +7, or later.

A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine.

Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34). The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34). I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34. We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon. Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC. Despite this, the mutant grows normally. As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced. However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC. Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon. We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG). We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA.

Influences on translation initiation and early elongation by the messenger RNA region flanking the initiation codon at the 3' side.

The downstream region (DR) located immediately after the initiation codon acts as a translational enhancer and depending on its sequence gene expression can vary considerably. In order to determine the influence of the DR on the apparent translation initiation, we have analyzed several naturally occurring DRs (a stretch of five codons) in a lacZ reporter gene. The efficiency of expression, associated with these DRs did not show any correlation to the expression levels connected with the natural genes. Changes of the iso-codon composition in the DR, thus maintaining the amino acid sequence in the gene product, gave significant variations in gene expression. Thus, the messenger RNA base sequence, and not the encoded amino acid sequence, in the early coding region is the determinant for the apparent efficiency of translation initiation and/or early elongation.

Functional EF-Tu with large C-terminal extensions in an E coli strain with a precise deletion of both chromosomal tuf genes.

An Escherichia coli strain was constructed in which both chromosomal genes encoding elongation factor (EF)-Tu (tufA and tufB) have been inactivated with precise coding sequence replacements. A tufA gene in an expression vector is supplied as the sole EF-Tu source. By using plasmid replacement, based on plasmid incompatibility, mutant EF-Tu variants with a large C'-terminal extension up to 270 amino acids were studied and proved to be functional in a strain lacking the chromosomal tufA and tufB genes.

Tandem termination signals: myth or reality?

In two Escherichia coli genomes, laboratory strain K-12 and pathological strain O157:H7, tandem termination codons as a group are slightly over-represented as termination signals. Individually however, they span the range of representations, over, as expected, or under, in one or both of the strains. In vivo, tandem termination codons do not make more efficient signals. The second codon can act as a backstop where readthrough of the first has occurred, but not at the expected efficiency. UGAUGA remains an enigma, highly over-represented, but with the second UGA a relatively inefficient back up stop codon.

A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli.

Uneven distribution of plasmid-based expression vectors to daughter cells during bacterial cell division results in an increasing proportion of plasmid free cells during growth. This is a major industrial problem leading to reduction of product yields and increased production costs during large-scale cultivation of vector-carrying bacteria. For this reason, a selection must be provided that kills the plasmid free cells. The most conventional method to obtain this desired selection is to insert some gene for antibiotic resistance in the plasmid and then grow the bacteria in the presence of the corresponding antibiotic. We describe here a host/plasmid Escherichia coli system with a totally stable plasmid that can be maintained without the use of antibiotic selection. The plasmid is maintained, since it carries the small essential gene infA (coding for translation initiation factor 1, IF1) in an E. coli strain that has been deleted for its chromosomal infA gene. As a result only plasmid carrying cells can grow, making the strain totally dependent on the maintenance of the plasmid. A selection based on antibiotics is thus not necessary during cultivation, and no antibiotic-resistance genes are present neither in the final strain nor in the final plasmid. Plasmid-free cells do not accumulate even after an extended period of continuous growth. Growth rates of the control and the plasmid harboring strains are indistinguishable from each other in both LB and defined media. The indicated approach can be used to modify existing production strains and plasmids to the described concept. The infA based plasmid stability system should eliminate industrial cultivation problems caused by the loss of expression vector and use of antibiotics in the cultivation medium. Also environmental problems caused by release of antibiotics and antibiotic resistance genes, that potentially can give horizontal gene transfer between bacterial populations, are eliminated.

Mutations in 16S rRNA that suppress cold-sensitive initiation factor 1 affect ribosomal subunit association.

A mutation in the infA gene encoding initiation factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site suppressors that compensate for the cold sensitivity and map specifically to rRNA. Several suppressor mutants with altered 16S rRNA that partially restore growth of an IF1 mutant strain in the cold were isolated and characterized. Suppressor mutations were found in helix (h)18, h32, h34 and h41 in 16S rRNA. These mutations are not clustered to any particular region in 16S rRNA and none overlap previously reported sites of interaction with IF1. While the isolated suppressors are structurally diverse, they are functionally related because all affect ribosomal subunit association in vivo. Furthermore, in vitro subunit-association experiments indicate that most of the suppressor mutations directly influence ribosomal subunit association even though none of these are confined to any of the known intersubunit bridges. These results are consistent with the model that IF1 is an rRNA chaperone that induces large-scale conformational changes in the small ribosomal subunit, and as a consequence modulates initiation of translation by affecting subunit association.

Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region.

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.

Translation initiation region dependency of translation initiation in Escherichia coli by IF1 and kasugamycin.

Translation initiation factor 1 (IF1) is an essential protein in prokaryotes. The nature of IF1 interactions with the mRNA during translation initiation on the ribosome remains unclear, even though the factor has several known functions, one of them being RNA chaperone activity. In this study, we analyzed translational gene expression in vivo in two cold-sensitive chromosomal mutant variants of IF1 with amino acid substitutions, R40D and R69L, using two different reporter gene systems. The strains with the mutant IF1 gave higher reporter gene expression than the control strain. The extent of this effect was dependent on the composition of the translation initiation region. The Shine-Dalgarno (SD) sequence, AU-rich elements upstream of the SD sequence and the region between the SD sequence and the initiation codon are important for the magnitude of this effect. The data suggest that the wild-type form of IF1 has a translation initiation region-dependent inhibitory effect on translation initiation. Kasugamycin is an antibiotic that blocks translation initiation. Addition of kasugamycin to growing wild-type cells increases reporter gene expression in a very similar way to the altered IF1, suggesting that the infA mutations and kasugamycin affect some related step in translation initiation. Genetic knockout of three proteins (YggJ, BipA, and CspA) that are known to interact with RNA causes partial suppression of the IF1-dependent cold sensitivity.

Influences on gene expression in vivo by a Shine-Dalgarno sequence.

The Shine-Dalgarno (SD+: 5'-AAGGAGG-3') sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon. The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site. The first start site is activated if the distance between this site and the downstream SD+ is enlarged and/or if the second start site is weakened. Upstream initiation is eliminated if a stable stem-loop structure is placed between this SD+ and the upstream start site. The results suggest that the two start sites compete for ribosomes that bind to an SD+ located between them. A minor positive contribution to upstream initiation resulting from 3' to 5' ribosomal diffusion along the mRNA is suggested. Analysis of the E. coli K12 genome suggests that the SD+ or SD-like sequences are systematically avoided in the early coding region suggesting an evolutionary significance.

Stepping transfer messenger RNA through the ribosome.

tmRNA (transfer messenger RNA) is a unique molecule used by all bacteria to rescue stalled ribosomes and to mark unfinished peptides with a specific degradation signal. tmRNA is recruited by arrested ribosomes in which it facilitates the translational switch from cellular mRNA to the mRNA part of tmRNA. Small protein B (SmpB) is a key partner for the trans-translation activity of tmRNA both in vivo and in vitro. It was shown that SmpB acts at the initiation step of the trans-translation process by facilitating tmRNA aminoacylation and binding to the ribosome. Little is known about the subsequent steps of trans-translation. Here we demonstrated the first example of an investigation of tmRNA.ribosome complexes at different stages of trans-translation. Our results show that the structural element at the position of tmRNA pseudoknot 3 remains intact during the translation of the mRNA module of tmRNA and that it is localized on the surface of the ribosome. At least one SmpB molecule remains bound to a ribosome.tmRNA complex isolated from the cell when translation is blocked at different positions within the mRNA part of tmRNA.