Termination of protein synthesis involves the recognition of one of three stop codons (UAG, UAA or UGA) and hydrolysis of the nascent polypeptide chain from the peptidyl-tRNA on the ribosome. Unlike sense codons, which are decoded by aminoacyl-tRNAs, stop codons are decoded by proteins known as release factors. The decoding release factors occupy the same site as aminoacyl-tRNA, interacting directly with the stop codon at the decoding centre and inducing peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Eubacteria have two codon-specific decoding release factors - RF1, which recognizes UAG and UAA, and RF2, which recognizes UGA and UAA. Biochemical studies identified two tripeptide 'anticodon' motifs, PXT in RF1 and SPF in RF2, which structural studies have shown occur in exposed loops (anticodon loops) on the surface of the proteins. Structures of isolated release factors show a compact 'closed' conformation whereas structures of release factors bound to the ribosome show them to be in a highly extended 'open' conformation. This suggests that a large conformational change in the release factor must take place upon or before binding to the ribosome. This transition has been invoked as a mechanism for how translational fidelity is maintained (Rawat et al, 2003), however, small angle X-ray scattering data from E. coli RF1 suggest the decoding release factors are also in the open conformation in solution challenging this mechanism.
Mora et al. (2003a) presented evidence that swapping the anticodon loop of RF2 with that of RF1 switched the stop codon specificity of the release factor. Recent structures of the decoding release factors bound to the ribosome showed that there was a second structural element of the release factor, the tip of helix α5, involved in recognition of the first base of the stop codon. The objectives of this thesis were to investigate both the anticodon loop and the helix α5 region for their roles in stop codon recognition, and to investigate whether there is a conformational change in the release factors on binding to the ribosome.
The anticodon loop was investigated using chimeras of E. coli RF1/RF2 and E. coli RF1/C. elegans mitochondrial RF1 (MRF1) within the anticodon loop. An RF1 variant containing the RF2-specific SPF tripeptide motif did not switch stop codon specificity showing that the tripeptide motifs are not sufficient determinants for the codon specificity of RF1 and RF2 as was originally proposed. Surprisingly repeating the complete swap of the RF1 anticodon loop to that of RF2 did not switch the stop codon specificity as claimed in Mora et al. (2003a). The studies in this thesis identified additional regions of the anticodon loop of the release factor that are important for stop codon recognition. Two of the RF1/RF2 anticodon loop variants produced showed altered codon specificity recognizing all three standard stop codons and the sense codon UGG. These variants provided unexpected insights into the mechanism of stop codon recognition and can explain why there are two release factors in eubacteria.
The C. elegans MRF1 contains a novel anticodon loop that is shorter and lacks the classical PXT motif. E. coli RF1/C. elegans MRF1 chimeras showed that this anticodon loop could function in E. coli RF1 and maintain the same codon specificity. While size and sequence within the loop together are important for recognition clearly there is more than one way RF1-type release factors can recognize the UAG and UAA stop codons.
Vertebrate mitochondria use four stop codons, two of the standard stop codons, UAA and UAG, and the reassigned arginine codons AGA and AGG. Two vertebrate mitochondrial release factors have been identified, mtRF1a and mtRF1 (renamed here mRF1[Canonical] and mRF1[Noncanonical]). Bioinformatic studies showed mRF1[C] had similar helix α5 and anticodon loop regions to classical RF1s. mRF1[NC] had different helix α5 and anticodon loop regions and was hypothesized to recognize the non-standard stop codons AGA and AGG. E. coli RF1/Human mRF1[NC] chimeras were constructed that showed that the helix α5 and anticodon loop regions are important for stop codon recognition. Nevertheless the chimeras showed poor activity at the AGA and AGG stop codons on E. coli 70S ribosomes suggesting that mRF1[NC] has evolved to function exclusively on 55S mitoribosomes.
A release factor variant of RF2 was designed that had the potential to trap this E. coli factor in the closed conformation in solution by disulphide bond formation. The RF2 double cysteine variant was successfully expressed and purified. The disulphide bond between the two cysteines was detected directly by mass spectrometry in a high proportion of molecules, showing the closed form of RF2 exists in solution. The RF2 closed form variant was shown to have release activity concomitant with the proportion of the open form in the RF preparation showing that the conformational change is required for normal release factor function. Preliminary binding studies have suggested that the RF2 closed form variant can bind to the ribosome. The ability of the closed form of RF2 to bind to the ribosome allowed a mechanism of translational fidelity to be proposed from the studies in this thesis; the release factor would recognize the stop codon in the decoding centre and, if cognate, the conformational change would occur allowing peptidyl-tRNA hydrolysis.
| Identifer | oai:union.ndltd.org:ADTP/234774 |
| Date | January 2009 |
| Creators | Young, David James, n/a |
| Publisher | University of Otago. Department of Biochemistry |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright David James Young |
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