RNA Journal Club 9/23/10
Hani S. Zaher & Rachel Green
Nature Vol 457, 8 January 2009.
This week’s cogent summary and analysis by Josh Arribere:
The authors initiate the paper with a discussion of known quality control mechanisms in protein synthesis. They present the overall rate in vivo as being in the range of 6e-4 and 5e-3, and state that their own in vitro measurements of fidelity are in the range of 1e-4 and 2e-3. From the overlap of these two ranges, it is not readily apparent that a new quality control mechanism need exist, but the true motivation for the study becomes apparent shortly. In the process of making an oligopeptide in vitro, the authors failed, and instead observed a miscoding event that led to premature termination.
It is from this observation the authors begin the paper. They demonstrate that although the rate of RF2-stimulated hydrolysis (release of the nascent peptide) is comparable for correct vs. miscoded events (fig 1c), the Km is ~10 fold less (fig 1d). Such a difference is surprising given the one base pair change between the two constructs. Furthermore, they demonstrate that the miscoded construct is subject to RF2-mediated release (increase in the rate of hydrolysis, fig 1f), albeit inefficiently, even when the A site lacks a stop codon. The Km is also decreased in the miscoded event, leading to an overall ~300 fold increase in the second order rate constant (fig S4). Different mismatch events and A-site codons argue that the observed phenomenon is not a peculiarity of their original construct (fig 1f). Moreover, of all the mismatches in the P-site, one is tolerated, namely, the G:U wobble base pair in the third position, as to be expected given the degenerate nature of the genetic code (fig S8). Thus the proposed mechanisms are compatible with known biology.
Primer extension assays demonstrate the ribosome has not shifted frame (fig S5), and although P-site tRNA dropoff is increased in the miscoded case, the rate of dropoff is ~2.5 fold slower than RF2-mediated release. 2.5 fold is a rather small gap, and the rate of RF2-mediated release is still two orders of magnitude slower than the rate of elongation. However, upon addition of RF3 (a class II release factor) and RF2, the rate of release increases another 10 fold (fig 2). This puts release following a miscoding event on par with the rate of chain termination, but still slower than elongation (~2/sec). So what happens if the tRNA beats the RFs to the ribosome? The rate of peptidyl transfer is not inhibited (fig 3a black bars), though the ribosome has ~10x diminished capacity to correctly incorporate the next amino acid following a miscoding event (fig 3a white bars). Examining the nature of the peptides formed reveals predominantly the correct tripeptide product in the correctly coded case. However, multiple seemingly random tripeptide products are formed following a P-site mismatch (fig 3b). Thus a single mismatch in the P site leads to a general loss of ribosome fidelity.
One of the consequences of these multiple miscoding events is a further stimulation of release by RF2 and RF3 (fig 4b,c,d). Peculiarly, an E-site mismatch alone does not stimulate release (except in the “buffer-dependent” instance of fig4b), but does stimulate release together with a P-site mismatch. This begs the question: how is an E-site mismatch only sensed together with a P-site mismatch and not by itself? Frame maintenance is somewhat compromised only in the doubly miscoded case (fig S13). Of interest, the only case where an E-site mismatch alone led to stimulation of hydrolysis (MNKF, fig 4b), also exhibits an abnormal primer-extension banding pattern similar to the doubly miscoded event (fig S13b, compare last two lanes). At any rate with this further RF2/3-stimulated increase in release for the doubly miscoded event, the rate of peptide hydrolysis (~1/sec) is now on par with elongation (~2/sec), making it a kinetically viable pathway in protein synthesis.
All of the above observations, together with the rate constants and concentrations of protein synthesis factors, are incorporated into a model (fig 5a). Testing the model with a S100 extract (supernatant of a 100,000g cell lysate) confirmed some of the predictions of the model. Following an initial miscoding event, the next correct amino acid is added ~30% of the time, and subsequently a relatively low loss of yield for this product is observed (fig 5c 3rd columns). Since the doubly miscoded event contains multiple species (many AAs possible), it is not possible to measure the “Incorrect PT” arrow in fig 5a with the TLC assay (and the authors note this). One confusing point is the apparent increase in MN-matched formation between the di- and tripeptide (fig 5c 2nd column of di-, tripeptide), though the authors do not comment on this.
The authors come an incredibly long way from a failed experiment (oligopeptide production) to discover proofreading by the ribosome. They keep an eye on rate constants to demonstrate the phenomena they are studying are kinetically relevant. Different in vitro translation labs each favor particular buffer systems (buffers A, B, C, D in this paper), and the authors quell arguments by repeating some of their observations in multiple buffer systems (for instance, fig S15). This is important since each buffer seems to have its own peculiarities (see fig 4b, S1, S3, S11), and it is not readily apparent what this means, nor which buffer, if any, is the “correct” one.
There are many future directions for further study, and some are mentioned in the paper. One that was not mentioned is the fate of the released peptide. A miscoded, truncated peptide is a potential dominant negative nightmare for the cell. Clearly there must be a tight coupling in the cell between peptide release and degradation. Subsequent unpublished experiments have shown that the mRNA is destabilized following miscoding, though I do not know about the fate of the nascent peptide. It would be very interesting to know what discerns miscoding and RF2/3 stimulated release from normal stop-codon mediated release.