Studies of the cause of random translational misreading errors
Because of our work on these programmed errors, we became interested in the question of how errors are regulated during protein synthesis more generally. The literature on random missense errors—those substituting one amino acid for another—mostly comes from several decades ago and the results of those studies are made more difficult to interpret because the data is sparse (only a few errors by any given tRNA had been studied) and contradictory. We felt it was important to revisit this topic and be as comprehensive as possible. In the last several years we have produced a series of papers that explore the phenomenon of misreading errors in bacteria and yeast; currently we are expanding that work to study these errors in human tissue culture cells.
The system we have used involves creating mutations that alter residues known to be directly involved in catalysis by a well-studied reporter protein (either Photinus pyralis, or firefly, luciferase, or Escherichia coli beta-galactosidase). The system allows us to quantify errors that result in the replacement of a mutant active site amino acid by the normal amino acid, restoring full enzyme activity to the reporter. These events result in a small proportion of total proteins encoded having full activity while the majority of correctly-translated but mutant proteins are either inactive or have very low activity. The frequency of misreading errors is equal to the increase in activity of proteins expressed by an error-prone mutant gene to the full activity produced by a wild type gene. For completeness, we test all possible errors by a given tRNA using these reporters, which means measuring the activity of all mutants that differ from wild type by a single nucleotide substitution. Such mutants are termed “near-cognate” as opposed to the wild type which has a correct or “cognate” codon. Mutants having more than one change from the wild type codon (“non-cognate” mutants) cannot be misread to produce wild type protein.
The results of these studies produced a few major conclusions.
- A given tRNA makes errors substantially above background on only a few codons.
- Errors are increased when the cognate tRNA specific for the mutant codon is in low abundance; this is because the error-inducing near-cognate tRNA must compete with this cognate codon to make the misreading error.
- The pattern of errors in the bacterium Escherichia coli, yeast Saccharomyces cerevisiae and human cells is quite similar with the same codons tending to cause errors in all three species. The differences in errors between species tend to reflect the abundance of the competing cognate tRNA. For example, the arginine codon AGA is read by a very low abundance tRNA in E. coli but a very abundant one in yeast. Errors by a lysyl-tRNA at AGA (by misreading the middle base of the codon) are relatively frequent in E. coli but not observed in yeast.
- The overall pattern of misreading is that errors are more frequent in bacteria than in yeast and that they are still less frequent in human cells. The reasons for this difference are complex but includes an increasing trend toward more similar concentrations of all cognate tRNAs in the eukaryotes.
By comparing errors by several tRNAs in bacteria and yeast we found a general rule that appears to explain why tRNAs make more errors at some codons than on others. The most frequent errors are those that involve specific nucleoside mismatches at one of the three codon positions. The most frequent are those that involve misreading of a G in the mRNA by a U in the tRNA (a G-U error). All such mismatches that we tested showed significant error frequencies. The next most frequent errors were those in which a U or C in the mRNA is paired with a U in the tRNA (U-U or C-U errors). All U-U or C-U errors in the third or “wobble” position of the codon showed significant error frequencies. No C-U errors at the other two codon positions showed observable errors but some U-U errors at the first or second position were frequent. A recent unbiased mass spectrometry experiment to measure misreading errors identified the same mismatches as causing the most frequent errors. Also, recent structure analysis has shown that G-U, U-U and C-U mismatches form pairs with geometry similar to the Watson-Crick (W-C) base pairs (G-C and A-U). Since the ribosome uses the dimensions (width) of base pairs to distinguish between correct, cognate W-C codon-anticodon complexes and all incorrect pairs, it appears that errors are most frequent with these mismatches because the ribosome cannot distinguish them from W-C pairs. Errors are infrequent mainly because these three mismatched pairs form infrequently and are fundamentally unstable, leading to their being rejected by the ribosome. However, because they are able to adopt a W-C conformation they are much more likely than any other mismatches to be accepted by the ribosome, resulting in increase error frequencies.
Current work in the laboratory is aimed at understanding how cellular physiology affects the absolute frequencies of errors. This includes the fact that the cell post-transcriptionally modifies bases in the tRNA anticodon loop which are either paired with the mRNA codon or are immediately adjacent and play a role in modulating codon-anticodon stability.
We are also interested in the relationship between accuracy and the structure of the ribosome, the complex ribonucleoprotein organelle responsible for performing translation. We are studying the effects of trans-acting factors that modulate error frequency including enzymes that post-translationally modify proteins in the ribosome that are known to be involved in translational accuracy. We are also interested in the relationship between the accuracy of protein synthesis and the correct assembly of the ribosome. We have recently published on the effect of deficits of individual ribosomal proteins on the accuracy and efficiency of translation and on the accuracy effects of lack of a late ribosomal assembly factor in bacteria, RbfA. These studies are continuing.