Fidelity at the Molecular Level: Lessons from Protein Synthesis
Protein translation is the process by which messenger RNA (mRNA) supplies the necessary information for the linear synthesis of proteins. There are three basic components to a cell's translational machinery: mRNA, tRNA, and ribosomes. Messenger RNA provides the template that will be used for ordering the correct sequence of amino acids. Fidelity of the translational process is assured, in part, by the fact that each amino acid has its own transfer RNA. Transfer RNA (tRNA) is found with an appropriate amino acid. For example, a tRNA that has an anticodon of "UAC" will bind to the triplet on the mRNA with the complimentary sequence "AUG." Thus, each tRNA delivers the appropriate amino acid to the ribosome; ordering of amino acids is determined by the linear arrangement of the genetic code. Be sure that you understand the relationship between these three components of the cell's translational machinery.
Solved: Protein synthesis occurs with high fidelity
This idea was later supported by the isolation of streptomycin-resistant (restrictive) mutations, as well as mutations that can suppress the streptomycin-dependent mutation phenotype and cause extensive miscoding (ribosomal ambiguity, ram). The restrictive mutations were ultimately mapped to genes encoding the small ribosomal protein S12 (rpsL), whereas the ram mutations were found to alter the small subunit ribosomal proteins S4 and S5 (rpsD and rpsE). These initial studies thus provided clear evidence that the ribosome controls the accuracy of decoding through multiple distinct loci, an idea now well supported by a great range of ram and restrictive mutations since identified in various ribosome components (reviewed in ). The mechanistic implications of these initial genetic clues are to a great extent revealed by current high-resolution structures (also discussed in ).
As mentioned, the process of transcription occurs in the nucleus of eukaryotic cells and requires that the two strands of DNA separate, or open up sufficiently enough so that complementary RNA nucleotides can be added to one side of the DNA molecule. The strand that is copied is the template strand. The enzyme RNA polymeraseseparates the DNA strands and joins the RNA nucleotides along the exposed DNA template. This process is initiated when certain proteins, transcription factors, bind to a specific starting point, the promoter. The promoter is actually a sequence of DNA bases that signals the beginning of RNA synthesis. RNA polymerase then adds nucleotides to the 3' end of the elongating RNA molecule. The enzyme then moves down the DNA strand, unwinding as it goes and allowing the DNA helix to reform after a sequence has been transcribed. This continues until a specific RNA sequence is transcribed. This sequence, the terminator sequence, signals the end of RNA synthesis. Transcription is broken down into three stages: initiation, elongation, and termination.
Protein synthesis or translation has an ..
In a relatively recent set of advances, the tRNA selection process has been studied using single-molecule Förster resonance energy transfer techniques (smFRET). The power of this approach comes from its inherent ability to follow individual behaviors in the dynamic multi-step pathway, especially those that are easily lost by averaging when studying the bulk properties of these same molecules. The feasibility of using these techniques for the study of protein synthesis is the result of recent methodological advances in the labeling, immobilization and detection of single ribosomal complexes (reviewed in ).
Fidelity in tRNA Aminoacylation ..
Early biochemical studies identified the decoding center of the ribosome to be located on the small 30S ribosomal subunit at the interface with the large 50S subunit, encompassing 16S rRNA nucleotides 1400–1500 of helix 44, the 530 loop (helix 18), and residues 1050–1500. Chemical modification protection analysis showed that the bases of the conserved nucleotides guanosine 529 (G529), guanosine 530 (G530), adenosine 1492 (A1492) and adenosine 1493 (A1493) are protected by the binding of an A-site tRNA (). Moreover, the aminoglycoside paromomycin induces protection of nucleotides 1408 and 1494, just across from 1492 and 1493 in helix 44 (). Mutational experiments later demonstrated that these nucleotides are critical for A-site tRNA binding (, ; ). Moreover, an NMR structure of an oligonucleotide corresponding to this region of helix 44 bound to paromomycin revealed that the aminoglycoside stabilized a structure of A1408, A1492 and A1493 that is distinct from that observed in the absence of ligand (; ). The authors suggested that the observed conformational changes might mimic those induced by the binding of cognate A-site tRNA to the ribosome.
the enzyme urease was a pure protein ..
It should be noted that overall selectivity is highly dependent on experimental conditions, with Mg2+ and polyamine concentrations playing an especially critical role (; ; ). Under reduced fidelity conditions, where Mg2+ is high and polyamines are absent, the initial selection stage is less effective, resulting in a relatively high error frequency (). Other in vitro studies using several different higher fidelity buffer systems have yielded misincorporation rates that more closely approach those reported in vivo (; ). These measurements are generally carried out by comparing the rate constants for one particular near-cognate tRNA with those of the cognate species and assuming that their concentrations are equal in vivo. As such, these calculations do not take into account that for each tRNA, multiple near-cognate tRNAs that compete with similar efficiencies also exist (). These near-cognant tRNAs will in turn increase the level of misincorporation by a factor dependent on their overall concentration. Recent in vivo experiments by Farabaugh and colleagues document the importance of tRNA competition in specifying the fidelity of tRNA selection (). Our in vitro experiments conducted with a complete competitor tRNA population mimicking the in vivo milieu yield somewhat higher error frequencies ranging from 2–10 misincorporations in 103 events ().