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, 8 (4), e60246

The Phenotype of Many Independently Isolated +1 Frameshift Suppressor Mutants Supports a Pivotal Role of the P-site in Reading Frame Maintenance


The Phenotype of Many Independently Isolated +1 Frameshift Suppressor Mutants Supports a Pivotal Role of the P-site in Reading Frame Maintenance

Gunilla Jäger et al. PLoS One.


The main features of translation are similar in all organisms on this planet and one important feature of it is the way the ribosome maintain the reading frame. We have earlier characterized several bacterial mutants defective in tRNA maturation and found that some of them correct a +1 frameshift mutation; i.e. such mutants possess an error in reading frame maintenance. Based on the analysis of the frameshifting phenotype of such mutants we proposed a pivotal role of the ribosomal grip of the peptidyl-tRNA to maintain the correct reading frame. To test the model in an unbiased way we first isolated many (467) independent mutants able to correct a +1 frameshift mutation and thereafter tested whether or not their frameshifting phenotypes were consistent with the model. These 467+1 frameshift suppressor mutants had alterations in 16 different loci of which 15 induced a defective tRNA by hypo- or hypermodifications or altering its primary sequence. All these alterations of tRNAs induce a frameshift error in the P-site to correct a +1 frameshift mutation consistent with the proposed model. Modifications next to and 3' of the anticodon (position 37), like 1-methylguanosine, are important for proper reading frame maintenance due to their interactions with components of the ribosomal P-site. Interestingly, two mutants had a defect in a locus (rpsI), which encodes ribosomal protein S9. The C-terminal of this protein contacts position 32-34 of the peptidyl-tRNA and is thus part of the P-site environment. The two rpsI mutants had a C-terminal truncated ribosomal protein S9 that destroys its interaction with the peptidyl-tRNA resulting in +1 shift in the reading frame. The isolation and characterization of the S9 mutants gave strong support of our model that the ribosomal grip of the peptidyl-tRNA is pivotal for the reading frame maintenance.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. The ribosomal grip of the peptidyl-tRNA is pivotal in reading frame maintenance.
The figure shows three ways (A, B and C) how certain events may induce slippage by the peptidyl-tRNA and thereby a frameshift error. It is the ternary complex (aa-tRNA*EfTu*GTP) which enters the A-site and interacts with the codon but in the figure we have symbolized it with “aa-tRNA” to save space. A. A defective cognate tRNA (red diamond) is slow (broken arrow) entering the A-site allowing a near-cognate aa-tRNA (blue wobble nucleoside) to decode the A-site codon. After a 3 nucleotide translocation the near-cognate peptidyl-tRNA may slip into the +1 frame. B. A defective cognate aa-tRNA (red diamond) decodes efficiently the codon in the A-site. After a 3 nucleotide translocation the defective cognate peptidyl-tRNA may be prone to slip into the +1 frame. C. The defective aa-tRNA (red diamond, yellow tRNA) is slow entering the A-site mediating a pause allowing the cognate wild type peptidyl-tRNA to slip into the +1 frame. Not depicted in the figure, alterations in the ribosomal P-site environment may also induce a frameshift error if the alteration changes the ribosomal grip of the peptidyl-tRNA. The figure is adopted from with permission. 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. 6). Moreover, the occupancy of the E-site also improves reading frame maintenance , –, 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.
Figure 2
Figure 2. Sequence and aminoacylation levels in vivo of various mutant :s.
The positions of charged (ch) Gln or Arg-tRNA and uncharged (unch) Gln-tRNA are indicated and their migration pattern was obtained from control experiments. The position of uncharged Arg-tRNA is between Gln-ch and Arg-ch as shown by a control experiment. Since Arg-tRNA was 100% charged the uncharged Arg-tRNA is not indicated in the figure. Sequence of wild type glnU formula image and various mutants (base alteration shown in red). s4U, 4-thiouridine, #, 2′-O-methylguanosine (Gm), D, dihydrouridine, J, 2′-O-methyluridine (Um), N, 5.-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), m2A, 2-methyladenosine, P, pseudouridine (Ψ), T, 5-methyluridine (m5U).
Figure 3
Figure 3. Amino acid sequence of the frameshift product encoded from plasmids pUST290, pUST292, pUST310, and pUST311.
The frameshift window, within which the frameshift must occur, is bordered by the stop codon UAA (italics and underlined) in +1 frame and the stop codon UGA UAA in the zero frame (Indicated by a * below the DNA sequence). P or F (in red) denote the last amino acid decoded in the zero frame found in the frameshift product.
Figure 4
Figure 4. Schematic picture of the synthesis of (c)mnm5s2U34, mnm5ges2U34, and se2(c)mnm5U.
(“ge” is a geranylgroup abbreviated “ge”; GPP is geranylpyrophosphate). The sulfur relay from Cys to the s2-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. A geranylgroup from GPP is transferred to cmnm5s2U of formula image by YbbB to generate the hypermodified ges2cmnm5U34 and to mnm5s2U of Lys- and Glu-tRNA to generate ges2 mnm5U . YbbB is also responsible for the exchange of s2 by Se forming mnm5Se2U if selenium phosphate is available .
Figure 5
Figure 5. Modified nucleosides in positions 32, 34, 37, and 38–40 and the coding capacities of the corresponding tRNAs.
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). proM tRNA has cmo5U34 as wobble base and decode all four proline codons . A circle corresponds to a codon read by a tRNA and the line between circles denotes that the same tRNA read those codons. Note also that the proM tRNA is essential, since it is the only tRNA reading the CCA codon. The proL tRNA having G34 as wobble nucleoside reads U and C ending codons and proK tRNA, which has C34 as wobble nucleoside, should read only CCG codon. The Gln codons CAA/G are read by two tRNAs having mnm5s2U34 and C34 as their wobble nucleoside. The C34 containing tRNA reads only CAG whereas the mnm5s2U containing tRNA (glnU tRNA) decodes both CAA and CAG although less efficient CAG (Unfilled circle). Note that the latter tRNA (glnU tRNA) is essential, since it is the only tRNA reading the CAA codon. In the Lys and Glu codon boxes one tRNA having mnm5s2U as wobble nucleoside reads AAA (Lys)/GAA (Gln) and less efficient AAG (Lys)/GAG (Glu) (Unfilled circle).
Figure 6
Figure 6. The anticodon loop of the peptidyl-tRNA and the extreme C-terminal end of ribosomal protein S9.
The anticodon of the peptidyl-tRNA is labeled blue, the C-terminal Arg130 of S9 is in purple and Lys129 is in orange. The arrow points to the 5′ phosphate of the wobble nucleotide pC34. The dashed line indicates a possible H-bond between the phosphate of pC34 and the amino group of Lys129 . The figure is adopted from with permission.

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    1. Woese CR (2002) On the evolution of cells. Proc Natl Acad Sci U S A 99: 8742–8747. - PMC - PubMed
    1. Parker J (1989) Errors and alternatives in reading the universal genetic code. Microbiological Reviews 53: 273–298. - PMC - PubMed
    1. Kramer EB, Farabaugh PJ (2007) The frequency of translational misreading errors in E. coli is largely determined by tRNA competition. RNA 13: 87–96. - PMC - PubMed
    1. Kramer EB, Vallabhaneni H, Mayer LM, Farabaugh PJ (2010) A comprehensive analysis of translational missense errors in the yeast Saccharomyces cerevisiae. RNA 16: 1797–1808. - PMC - PubMed
    1. Kurland CG (1992) Translational accuracy and the fitness of bacteria. Annu Rev Genet 26: 29–50. - PubMed

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Grant support

This work was supported by the Swedish Science Research Council (BU-2930), The Carl Trygger Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.