Trimethoprim TMP is an antibiotic that can bind to the active site of the chromosomal DHFR to inhibit the synthesis of tetrahydrofolate in bacteria. As shown in Figure 4B and C , all three proteins allowed E. To obtain the pure frame-shifted proteins, we changed the stop codons TAG and TGA into the code of one common amino acid in turn, and finally screened out the active mutants without internal stop codon.
When the WT DfrB3 was analyzed under the identical condition, a peak at Solid lines show best fit to the Michaelis—Menten equation. As shown in Figure 4G and Supplementary Figure S23 , the signals were assigned to the respective entities by ESI-MS analysis, and much to our delight we observed the disappearance of the signal of dihydrofolate, as well as the appearance of a signal of tetrahydrofolate in the reaction mixture, which is consistent with the catalytic result of WT DfrB3 protein Figure 4G and Supplementary Figure S However, the catalytic efficiency of three enzymes is apparently different from each other according to the fluorescence-tracing results Supplementary Figure S This result might explain why the WT dfrB3 gene is preserved in bacteria, while the other two frameshift genes are hidden.
A textbook gene encodes a protein using a single reading frame. In , Ohno discovered a protein in Flavobacteria with the capacity to degrade nylon, and this protein is coded within a previously existing gene, but in an alternative reading frame Although an experimental verification of two protein-coding genes in the same DNA locus is extremely challenging, an increasing number of nontrivially overlapping genes in prokaryotes have been found over the last few decades The frameshift mutation is generally considered to be lethal because it could result in radical changes of the chemical composition of the protein sequence behind.
On one hand, the unexpected results mentioned above is totally out of our imagination according to the existing knowledge, but on the other hand, from an evolutionary point of view, the observations might seem reasonable.
As a central pillar of the modern evolutionary theory, all organisms now living on the earth are believed to have a last universal common ancestry LUCA , which was probably highly error prone to evolve into different species. Thus, if the proteins encoded by a given gene from different reading frames have the same function, LUCA could effectively resist the instability caused by the frameshift mutations in reproduction, especially those genes that are crucial to the survival of LUCA and its descendants.
As described in our research, both TA systems and the synthesis of tetrahydrofolate are essential for the survival of bacteria under stressed conditions. As organisms continue to become more refined and precise during evolution, the protein with the best catalytic efficiency could finally win out, and other protein-coding sequences from different reading frames would be hidden consequently.
A possible way to find out those hidden genes is proposed by using the absence of extra TAA in ORF as a clue, because internal TAA means that the frame-shifted protein has no function at all, or their function is no longer needed in any case. Based on this hypothesis, we indeed found new hidden genes, but it would face enormous challenges because the percentages of genes without internal TAA varies greatly in different bacteria, such as 1.
Furthermore, although certain frame-shifted proteins in prokaryotes were verified to retain the same function as the WT protein here, whether the described phenomenon exists in eukaryotes needs to be questioned and explored. Page R. Toxin-antitoxin systems in bacterial growth arrest and persistence. Google Scholar. Masachis S. Type I toxin-antitoxin systems: regulating toxin expression via shine-dalgarno sequence sequestration and small RNA binding.
Fozo E. Gerdes K. Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. EMBO J. Kawano M. Molecular characterization of long direct repeat LDR sequences expressing a stable mRNA encoding for a amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli.
Unoson C. A small SOS-induced toxin is targeted against the inner membrane in Escherichia coli. Berghoff B. RNA-based regulation in type I toxin-antitoxin systems and its implication for bacterial persistence. Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells. Ramage H. Comprehensive functional analysis of Mycobacterium tuberculosis toxin-antitoxin systems: implications for pathogenesis, stress responses, and evolution.
PLoS Genet. Pandey D. Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes.
Nucleic Acids Res. Seligmann H. The ambush hypothesis: hidden stop codons prevent off-frame gene reading. DNA Cell Biol. Mok W. A highly efficient molecular cloning platform that utilises a small bacterial toxin gene. Abundance of type I toxin-antitoxin systems in bacteria: searches for new candidates and discovery of novel families.
Decoding toxicity: deducing the sequence requirements of IbsC, a type I toxin in Escherichia coli. Huang X. Intracellular selection of trans-cleaving hammerhead ribozymes. Buckingham R. Codon context. Cridge A. Wickens H. Flow cytometric investigation of filamentation, membrane patency, and membrane potential in Escherichia coli following ciprofloxacin exposure. Agents Chemother. Vogel J. Weel-Sneve R.
Single transmembrane peptide DinQ modulates membrane-dependent activities. Loh S. Nucleotide sequence and transcriptional analysis of a third function Flm involved in F-plasmid maintenance. Nielsen A. Mechanism of post-segregational killing by hok-homologue pnd of plasmid R two translational control elements in the pnd mRNA. Wang X. Radstrom P. Transposon Tn of plasmid R, which carries an integron, is related to Tn7, Mu, and the retroelements.
Schnell J. Structure, dynamics, and catalytic function of dihydrofolate reductase. Sanger F. The nucleotide sequence of bacteriophage phiX Indeed, as shown in this paper a truncation of ribosomal protein S9, which interacts with the peptidyl-tRNA induces an error in reading frame maintenance See Fig.
Moreover, the occupancy of the E-site also improves reading frame maintenance [80] , [86] — [88] , perhaps by strengthening the ribosomal grip of the peptidyl-tRNA. Therefore, a defective tRNA may also increase frameshifting by altering the dissociation rate of it from the E-site. An unbiased test of the proposed model Fig. According to the model we expected that many loci would, in some way, influence the structure and the activity of a tRNA.
Interestingly, two mutants had a defect in a loci not altering any tRNA but changing the P-site environment. The isolation and characterization of these rpsI mutants gave a strong support of our model that the ribosomal grip of the peptidyl-tRNA is pivotal for the reading frame maintenance. The bacterial strains used were derivatives of Salmonella enterica serovar typhimurium and Escherichia coli Table 1. As rich media Luria-Bertani LB was used [42]. The minimal solid medium was made from the basal medium [43] with 15g of agar per liter and supplemented with 0.
TYS-agar 10g of Trypticase Peptone, 5g of yeast extract, 5g of NaCl, and 15g of agar per liter was used as solid rich medium. The hisC mutation was used earlier to obtain several of the classical frameshift suppressor mutants [46] and apparently only very small amount of the HisC enzyme is required to enable a mutant to form colonies within a day or two without histidine in the growth medium.
We also introduced other frameshift mutations at the same site as in hisC to widen our possibilities to obtain various frameshift suppressor mutants. Thus, only a fraction of a percent suppression of a frameshift mutation in the hisD gene is required to make enough of a functional HisD enzyme to allow a colony to appear within a few days.
Monitoring suppression of a frameshift mutation in the hisD gene would be a sensitive way to detect mutations mediating very weak suppressor activity.
This latter strain was transformed with a 60 nt DNA oligonucleotide designed to replace the tetracycline resistance cassette with the designed frameshift mutation selecting tetracycline-sensitive recombinants [48] ; e. The complete frameshift windows used in this study are listed in Table 2. The construct was conjugated into S. To mutagenize with hydroxylamine a phage P22 lysate prepared from strain GT [pool of random Tn 10d Tc insertions in strain GT containing a deletion of the hisD and hisC genes his , thus avoiding recombination with wild type his -operon in transductions ] was treated with hydroxylamine as described [53] until approximately 0.
Around the NG crystal a ring of growing bacteria emerged that contains bacteria with mutations induced by NG. Bacteria were scraped off from several fractions of the bacterial ring and resuspended in 1 ml of LB and allowed to grow for 1—2 hours before phage P22 was added to make lysates.
To avoid siblings only one mutant from each phage stock was saved. Tet R clones were selected and His - clones were screened. A set of mobile plasmids containing most of the ORFs from E. If the plasmid reversed the suppressor phenotype to the wild type phenotype i.
This was verified by determining the DNA sequence of the mutated gene on the chromosome. Following centrifugation the pellet was suspended in 1 ml of ice-cold water. Following centrifugation, the supernatant was transferred to a new tube and RNA was precipitated by adding 3 volumes of ethanol. Half of the sample was diluted with equal volume of 0.
RNA was transferred to Zeta probe membrane and was detected as above. The column was washed once with 6 ml R and once with 2. Finally, tRNA was eluted with 7 ml R, precipitated by 0. To monitor ribosomal slippage and to purify the slippage product, a previously described system was used [58] , [59]. The bands corresponding to the MBP-His 6 peptides were excised from the membrane and subjected to N-terminal sequence analysis by Edman degradation.
Unmodified cysteine is too reactive during N-terminal sequencing and is usually only seen indirectly as the absence of an amino acid in one cycle. The confirmation of the presence of cysteine has been described earlier [36]. The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN.
The LSID for this publication is: urn:lsid:zoobank. According to our frameshift model [16] , [36] the shift in frame occurs not by an error in the A-site as e.
There are several ways that may induce a shift in frame see Introduction. These were, after the initial selection, subjected to a careful analysis of their frameshift suppressing phenotype. Such an analysis would reveal whether or not their frameshifting phenotypes were consistent with the model. Furthermore, we placed the frameshift mutations both the hisC and hisD derivatives on the chromosome not on a plasmid! Therefore, we expected these systems to be good tools to extensively test our model.
Alternatively, we mutagenized cultures of strains harboring one of the indicated his -mutations or a his deletion by nitrosoguanine NG. Phage P22 were grown on such cultures and used to infect strains having one of the his -mutations shown in Table 2. Care was taken to avoid siblings by saving only one unique mutant from each phage stock. Thus, all mutants characterized Table 3 are of independent origin even if the mutation resulted in the same nucleotide substitution.
The location of the transposon on the chromosome was determined by DNA sequencing out from the transposon and into the nearby chromosomal region. A glutamine codon is present in two of the six frameshift sites used Table 2.
We therefore expected mutations influencing the activity of and indeed this was the case. We obtained 93 mutants as extragenic suppressors in the glnU gene to the hisD mutation based on their linkage to a transposon close to the structural gene glnU for.
Of these 11 were verified by determination of the glnU sequence. We also obtained 13 mutants as extragenic suppressors to hisC , which were sequenced and further analyzed Table 4 , Fig. Three of these mutants displayed a temperature sensitive phenotype glnU, , nine glnU; ; were cold sensitive, and one mutant glnU had a reduced growth which was similar at all three temperatures tested Table 4.
According to our model Fig. Transfer RNA was prepared from wild type and from the various mutants under conditions which preserve the aminoacylation of tRNA [62]. Sequence of wild type glnU and various mutants base alteration shown in red.
For that purpose the plasmid pUST was constructed Fig. This protease cuts the protein at the specific protease site between the GST moiety and the rest of the peptide. P or F in red denote the last amino acid decoded in the zero frame found in the frameshift product.
The sequence of this frameshift peptide revealed that it was the wild type in the P-site that caused the frameshifting event Fig. Thus, in both cases low concentration of the Gln- caused the wild type peptidyl-tRNA Pro or Phe to slip forward one nucleotide and thereby moving the ribosome into the zero frame.
Since both wild type Pro- and Phe-peptidyl tRNA, which interact with two different codons in the P-site were induced to slip by a ribosomal pause, the identity of the last amino acid in the peptiyl-tRNA and the anticodon-codon interaction in the P-site is not critical. Thus, the frameshift phenotype of these mutants was consistent with our model Fig. Since this is an essential gene it was not surprising that we only obtained one mutant.
The first step in the synthesis of the side chain present at position 5 of wobble nucleoside c mnm 5 s 2 U34, which is present in tRNAs specific for Gln, Lys and Glu, is catalyzed by a heterodimer of MnmG earlier denoted GidA and MnmE proteins [64] Fig.
This reaction generates the cmnm 5 -side chain in the presence of glycine or nm 5 -side chain in the presence of ammonia. The MnmC1 activity of MnmC MnmC enzyme contains two activities, C1 and C2 [65] converts the cmnm 5 -group to an nm 5 -group which in turn is converted to the mnm 5 -side chain by the MnmC2 methyltransferase activity.
The cmnm 5 -side chain is present in a subset of whereas and chains contain only the mnm 5 -side chain. Therefore, the synthesis of the mnm 5 -side chain depends on four enzymatic activities and any alterations of these proteins encoded by the mnmE , mnmG and mnmC genes might change the extent of modification of the wobble nucleoside c mnm 5 s 2 U34 and thereby inducing inefficient decoding.
The sulfur relay from Cys to the s 2 -group of the nucleoside is shown in red and the different enzymes involved in the synthesis of these thiolated derivatives are shown in green denoted as protein with their genetic symbols starting with a capital letter. YbbB is also responsible for the exchange of s 2 by Se forming mnm 5 Se 2 U if selenium phosphate is available [89].
The first step in the synthesis of the s 2 -group, also present in mnm 5 s 2 U, is catalyzed by the cysteine desulfurase IscS, whose activity is required for the synthesis of all thiolated nucleosides in bacteria [66] , [67]. Deficiency of the s 2 -group should negatively influence the coding capacity of the three tRNAs having c mnm 5 s 2 U34 as wobble nucleoside. This large fraction of this kind of mutants depends on the fact that 10 genes are the targets for mutations reducing the synthesis of the c mnm 5 s 2 U Several of these mutants were analyzed for the level of c mnm 5 s 2 U in their tRNA.
Both the frameshifting phenotype and the reduced level of c mnm 5 s 2 U in the tRNA were returned to that of the wild type by introducing a complementing plasmid Table 5.
According to our model, reduced activity of Gln- due to deficiency of the modified nucleoside should induce a shift in frame by the peptidyl-Pro-tRNA and indeed this is the case [16]. Moreover, the entry of , which also contains the wobble nucleoside mnm 5 s 2 U, to its cognate codon AAG in the A-site should also be reduced.
Thus, we conclude that the frameshifting phenotype of these 72 mutants lacking c mnm 5 s 2 U34 is consistent with our proposed model Fig. One mutant was found to have an altered YbbB protein G67R.
This generates the presence of mnm 5 ges 2 U34 in a fraction of which in turn reduces the level of glutaminylated. Interestingly, the alteration of YbbB found here ybbB ,G67R is an amino acid substitution at the same position of YbbB as a mutant denoted ybbB ,G67E isolated in and earlier characterized by us [69]. A reduced level of charged induces a pause and allows the peptidyl-Pro- to shift frame.
The resulting frameshift peptide is consistent with this interpretation [69]. Thus, the frameshifting phenotype of the YbbB G67R mutant was found to be consistent with the proposed model Fig. Such reduced rate of A-site selection results in a frameshift in the P-site [71].
We characterized 10 hisT mutants and found that their frameshifting phenotype is consistent with our model Fig. The four proline codons are read by proL , proM and proK and their coding capacities are shown in Figure 5.
Both these frameshift suppressors have an extra base inserted in the anticodon loop [34] , [74]. Table 6 shows that deletions, duplications, base substitutions and promoter mutations were obtained.
However, the proK is not essential [73] and we expected an equally large amount of mutations in this gene as in proL. However, this was not the case and moreover, the two independently isolated proK mutants have an insertion of a G in the anticodon similar to the classical sufA6 tRNA Pro [34].
The small number of proK mutants suggests that only specific alterations of the proK gene induce frameshifting and this aspect is discussed below See Discussion.
In the proline coding box there are three tRNAs reading the four proline codons and they are encoded by proK , proL , and proM One copy of each gene is present in Salmonella. A circle corresponds to a codon read by a tRNA and the line between circles denotes that the same tRNA read those codons. For proL mutants it has been shown earlier that the frameshift occurs in the P-site [35] and this is the case also for mutants in proK [34] and in proM [36].
Thus, the frameshifting phenotypes of all these mutants defective in any of the three tRNA Pro are all consistent with the frameshifting model [ Fig. We obtained 19 trmD mutants of which 6 were sequenced and tested for the level of m 1 G in their tRNAs. Lack of m 1 G37 was the first modification deficiency shown to induce frameshift errors [18] and such deficiency induces frameshift errors in the P-site [35] consistent with our model Fig.
Thus, ribosomal protein S9 might be an important feature of the ribosomal grip of the peptidyl-tRNA in order to maintain the reading frame. Analysis of these mutants as well as two mutants isolated by direct substitution of amino acid R and K revealed that the frameshift occurred in the P-site as shown both by peptide sequencing of the frameshift product and by reduced frameshift by overexpression of the aa-tRNA predicted to read the A-site codon [36].
Arginine arg has been replaced by glutamate glu. See Figure 2 for the amino acid code. The addition or deletion of the nucleotides in the multiples of three, however, will not alter the reading frame. Thus, the protein in such cases would likely have either an extra or missing amino acid.
Usually, frameshift mutations occur as caused by a mutational error during DNA repair or replication. They can also occur by exposure to acridine dyes, which are capable of inducing frameshift mutations.
Due to insertion or deletion also referred to as indels of the nucleotide, the reading frame of the nucleotide sequence changes; however, the implication of these mutations depends on where they occur. The addition or deletion of a nucleotide can occur at the interstitial or intercalary position.
In some instances, the addition and the deletion of nucleotides occur simultaneously known as double frameshift , which eventually restore the reading frame to normal.
The outcome of the frameshift mutation may be a complete loss of protein structure and functionality, resulting in the non-functional polypeptide. However, the effect of the mutation at the phenotypic level will be determined by the resulting codons, post -mutation, and the mutation position.
Hence, frameshift mutations result in an abnormal or defective protein product containing an improper sequence of amino acids. Depending upon the location of the mutation, such proteins may be wholly new or non-usable. Frameshift mutation can also result in the stop codon. This occurrence of the premature stop codon on mRNA will terminate the translation process, thereby, resulting in a short-length polypeptide.
Depending on the extent and nature of frameshift mutation, the protein may either be shorter or longer in comparison to the normal protein. Such mutation can occur either spontaneously or due to environmental stimuli. An interesting fact is that frameshift mutations generally occur in the Adenine-Thymine AT -rich regions of the nucleic acid. Frameshift mutations can occur either by deleting or inserting the nucleotide in the nucleic acid Figure 3.
Deletion frameshift mutation , wherein one or more nucleotides are deleted in a nucleic acid, resulting in the alteration of the reading frame, i. Deletion is a more common mechanism for inducing the frameshift mutation that results in an altered reading frame.
Insertion frameshift mutation , wherein one or more nucleotides are added to the base sequence of the nucleic acid, which results in the change in the reading frame. The severity of this type of frameshift mutation is dependent on the number of nucleotides and the position of insertion of nucleotides. This mutation is also referred to as - 1 frameshift mutation.
This genetic code is present as a three nucleotides sequence. Each triplet of the nucleotide is eventually translated to form specific proteins required for various life processes. The conversion of this genetic code to protein occurs in two essential steps Figure 4. The transmission of genetic traits in initial genetic experiments by Gregor Mendel indicated that genetic information is carried from one generation to another in some discrete physical and chemical entity.
Later, amino acids were thought to be the carriers of genetic information. Marshall Nirenberg, Heinrich J. Matthaei, and Har Gobind Khorana revealed the nature of a codon and deciphered the codons. The whole-genome sequence is divided into consecutive, non-overlapping sequences of three nucleotides.
The triplet codon that initiates the translation process defines the reading frame. Each triplet of the nucleotide encodes a specific amino acid or a stop signal known as a codon. There are 64 codon combinations that encode 20 amino acids. However, out of these 64 codons, three are the stop codons; thus, 61 codons code for amino acids and three codons for the termination of the translation process i. Each codon is translated from an mRNA to an amino acid.
These amino acids are then joined together by the ribosomes in a process known as ribosome translocation. Synthesis of protein is a cyclic process wherein, after joining one amino acid to the growing chain of the polypeptide, the ribosome moves forward by three bases i. The movement of ribosomes has disproportionate effects on protein or polypeptide function. In case mutation occurs in the above sequence and an A nucleotide is added or inserted after the start codon AUG.
0コメント