1. In cells, in a variety of biochemical processes, when something goes havoc for some reason, there is readily an armada of different error check and repair mechanisms with their "antenna" out to detect errors, and correct them, preventing lethal consequences.
2. Leaking Cells membranes need to be fixed. During DNA replication, and translation, error check and repair is essential. Cells are endowed with a wide variety of specialized DNA repair mechanisms to counteract daily attacks: base excision repair, nucleotide excision repair, homologous recombination repair, mismatch repair, photoreactivation, nonhomologous end joining, translesion synthesis, and processing by the MRN complex. The Ribosome alone has 13 different error-check and repair mechanisms. In addition to repairing damage to existing DNA, living organisms have mechanisms to correct errors during reproduction. Bacteria have three types of DNA polymerase, all capable of detecting an incorrect base pairing, backing up one step to excise the incorrect nucleotide, and then progressing forward in a process called proofreading. The proofreading step decreases the error rate in bacteria from approximately one error in 100,000 base pairs to one error in 10,000,000 base pairs.
3. Molecules don't care if they are assembled in a way to bear a specific function. And if they do and the function is damaged and breaks down, those molecules neither "care" that they cease bearing that function.
4. Know how to implement an error check and repair system requires foresight. The very concepts of proofreading and repair implies goal orientation and "know-how" to keep something working and going. Those things can only come from an intelligent agency which implements these systems for specific purposes.
Molecules don't care if they are assembled in a way to bear a specific function. And if they do and the function is damaged and breaks down, those molecules neither "care" that they cease bearing that function. The very concepts of proofreading and repair implies goal orientation and "know-how" to keep something working and going. In cells, in a variety of biochemical processes, when something goes havoc for some reason, there is readily an armada of different error check and repair mechanisms with their "antenna" out to detect errors, and correct them, preventing lethal consequences . How is this not evidence of intended implementation by intelligence to achieve the specific purpose to maintain the complex cell machinery intact and operating properly? In some cases, very complex multi-part machines are involved performing efficient action to keep other complex molecular machinery and systems outside their own structural needs functional. They operate like fire-men that are called to cease a fire and rebuild the damaged part of the house ( photosystem II). Or a computer technician is called to repair a hard disk ( DNA ) Or a mechanic that is called to repair a 3D printer ( ribosome). But in contrast to human intervention, this biomolecular machinery is preprogrammed to know beforehand exactly when something is not working properly, and is able to work like a roboter and with the precision similar to a surgeon.
The process of gene expression is critical to all life-forms. No life-form could/would survive without these advanced mechanisms in place right from the beginning, when life started. In the pathway from Genes to proteins, and even post-translation, there are many sources of errors - and they must be fixed. Erroneous protein synthesis is due to any disruption in the conversion of the informational nucleotide sequence stored in DNA into a functioning protein. Besides amino-acid misincorporations, sources of errors are transcription errors, aberrant splicing, premature termination, faulty posttranslational modifications, and kinetic missteps during folding. This definition explicitly includes correctly synthesized polypeptides that fail to fold into a functional protein.Translation is the most error-prone step of protein synthesis.
The act of anticipation — foresight — is not a characteristic of blind material processes. It is an act of intelligence, of a mind.
Translation is the most error-prone step of protein synthesis. 14
The control of the error rate and its effects in biological processes of information transmission is one of the key requirements to have functional living cells.
When talking about error check and repair of translation in the cell, then we have to consider several aspects. First, in order to have a functional and error-prone translation, the components involved in translation must be correctly synthesized in the cell. In translation, messenger RNA, transfer RNA, Aminoacyl-tRNA synthetase, and the Ribosome are involved. Most, if not all, are carefully error checked, either repaired when errors are detected, or discarded when misfolding through proteostasis.
The process of translation is a multistep process, and error check and repair occur all along the way.
Kyle Mohler Translational fidelity and mistranslation in the cellular response to stress 2017 Nov 21.
Mistakes during DNA replication are on the order of ~10^8 and are kept to this extremely low level by a robust suite of error prevention, correction and repair mechanisms. Protein synthesis, offers the greatest opportunity for errors, with mistranslation events routinely occurring at a frequency of ~1 per 10,000 mRNA codons translated. The ribosome must select the correct aminoacyl-transfer RNAs (aa-tRNAs) from a large pool of near-cognate substrates fast enough to sustain an elongation rate of 10–20 amino acids per second !!. Proofreading and editing processes are used throughout protein synthesis to ensure a faithful translation of genetic information. The maturation of tRNAs and mRNAs is monitored, as is the identity of amino acids attached to tRNAs. Accuracy is further enhanced during the selection of aminoacyl-tRNAs on the ribosome and their base-pairing with mRNA.
In addition to misactivation of genetically encoded proteinogenic amino acids (GPAs), cells also encounter non-proteinogenic amino acids (NPAs) environmentally or as metabolic by-products, and must discriminate against these substrates to prevent aberrant use in protein synthesis. This is a list of eleven different error check and repair mechanisms during translation. Consider, that life cannot start, unless these mechanisms are fully in place, and operational. Consider as well, that all this machinery is a pre-requirement for living cells to kick-start life. Their origin cannot be explained by evolution. The alternatives are either all these hypercomplex life essential error check and repair mechanisms emerged by a fortuitous accident, spontaneously through self-organization by unguided stochastic coincidence, natural events that turned into self-organization in an orderly manner without external direction, chemical non-biological, purely physico-dynamic kinetic processes and reactions influenced by environmental parameters, or through the direct intervention, creative force and activity of an intelligent agency, a powerful creator. Which of the two makes more sense?
Ribosome biogenesis: quality control mechanisms must be in place to survey nascent ribosomes and ensure their functionality.
1. Chiral checkpoints during protein biosynthesis the ribosome act as “chiral checkpoints” by preferentially binding to L-amino acids or L-aminoacyl-tRNAs, thereby excluding D-amino acids 11
2. If misaminoacylated tRNA is successfully delivered to the ribosome, additional proofreading occurs within the A site of the ribosome based on aa-tRNA position and affinity
Ribosomal interactions with additional tRNA-specific sequences and modifications facilitate accurate selection of aa-tRNAs based on kinetic discrimination during the initial selection stage and subsequent proofreading stage.
3. mRNA translation regulation by nearly 100 epigenetic tRNA modifications. The finer details in this sort of regulation have been proven to differ between prokaryotic and eukaryotic organisms. ( LUCA hello ??!! 10)
4. tRNA structure monitoring: Export of defective or immature tRNAs is avoided by monitoring both structure and function of tRNAs in the nucleus, and only tRNAs with mature 5′ and 3′ ends are exported. 6
5. tRNA synthesis quality control: RTD and other mechanisms that degrade hypomodified or mutated mature yeast tRNAs serve as a surveillance system to eliminate tRNA molecules that have incorrect nucleosides or conformations
6. Aminoacyl-tRNA synthetase error minimization by preferential binding of the right amino acids, and selective editing and proofreading of near cognate amino acids
7. Aminoacyl-tRNA synthetase Pre-transfer editing: Pre-transfer editing has been described in both class I and class II aaRSs and takes place after aa-AMP synthesis but before the aminoacyl moiety is transferred to the tRNA. 9
8. Aminoacyl-tRNA synthetase Post-transfer editing: Post-transfer editing takes place after the transfer of the amino acid to the tRNA and involves the hydrolysis of the ester bond, in a domain separated from the active site.
9. Aminoacyl-tRNA synthetase Editing factors: Another important component of the translation quality control machinery is the trans-editing family, free-standing proteins that are not synthetases but are in some cases homologs to the editing domains of such enzymes. The role of these trans-editing factors is to clear the misacylated tRNA before it reaches the ribosome, acting as additional checkpoints to ensure fidelity.
10. Aminoacyl-tRNA synthetases (aaRSs), selectively hydrolyze ( chemical reaction in which a molecule of water ruptures one or more chemical bonds ) incorrectly activated non-cognate amino acids and/or misaminoacylated tRNAs. 12
B.Alberts Molecular Biology of the Cell. 4th edition: From RNA to Protein 2002
Several mechanisms working together ensure that the tRNA synthetase links the correct amino acid to each tRNA. The synthetase must first select the correct amino acid, and most synthetases do so by a two-step mechanism. First, the correct amino acid has the highest affinity for the active-site pocket of its synthetase and is therefore favored over the other 19. In particular, amino acids larger than the correct one are effectively excluded from the active site. However, accurate discrimination between two similar amino acids, such as isoleucine and valine (which differ by only a methyl group), is very difficult to achieve by a one-step recognition mechanism. A second discrimination step occurs after the amino acid has been covalently linked to AMP. When tRNA binds the synthetase, it tries to force the amino acid into a second pocket in the synthetase, the precise dimensions of which exclude the correct amino acid but allow access by closely related amino acids. Once an amino acid enters this editing pocket, it is hydrolyzed from the AMP (or from the tRNA itself if the aminoacyl-tRNA bond has already formed), and is released from the enzyme. This hydrolytic editing, which is analogous to the exonucleolytic proofreading by DNA polymerases , raises the overall accuracy of tRNA charging to approximately one mistake in 40,000 couplings.
Editing significantly decreases the frequency of errors and is important for translational quality control, and many details of the various editing mechanisms and their effect on different cellular systems are now starting to emerge. 8
Aminoacyl-tRNA synthetases must perform their tasks with high accuracy. Every mistake they make will result in a misplaced amino acid when new proteins are constructed. These enzymes make about one mistake in 10,000. For most amino acids, this level of accuracy is not too difficult to achieve. Most of the amino acids are quite different from one another, and, as mentioned before, many parts of the different tRNA are used for accurate recognition. But in a few cases, it is difficult to choose just the right amino acids and these enzymes must resort to special techniques.
Miguel Angel Rubio Gomez: Aminoacyl-tRNA Synthetases April 17, 2020
Isoleucine is a particularly difficult example. It is recognized by an isoleucine-shaped hole in the enzyme, which is too small to fit larger amino acids like methionine and phenylalanine, and too hydrophobic to bind anything with polar sidechains. But, the slightly smaller amino acid valine, different by only a single methyl group, also fits nicely into this pocket, binding instead of isoleucine in about 1 in 150 times. This is far too many errors, so corrective steps must be taken. Isoleucyl-tRNA synthetase (PDB entry 1ffy) solves this problem with a second active site, which performs an editing reaction. Isoleucine does not fit into this site, but errant valine does. The mistake is then cleaved away, leaving the tRNA ready for a properly-placed leucine amino acid. This proofreading step improves the overall error rate to about 1 in 3,000. 9
My comment: This is an amazing error proofreading technique, which adds to other repair mechanisms in the cell. Once again the question arises: How could these precise molecular machines have arisen by natural means, without intelligence involved? This seems to be one more amazing example of highly sophisticated nanomolecular machinery designed to fulfill its task with a high degree of fidelity and error minimization, which can arise only by the foresight of an incredibly intelligent creator.
I. Why wasn’t one ancestor enough when they both do the same job?
II. How did the two types of ancestral synthetases avoid competition that might have eliminated the inferior Class? 12
Errors that occur during transcription and translation have substantial effects on gene function by producing misfolded and malfunctioning proteins. The rate of translation errors is typically an order of magnitude higher than the rate of transcription errors. 1 Errors occurring during transcription often elicit more dire consequences than those occurring during translation because individual mRNAs can be translated up to 40 times resulting in a burst of flawed proteins. Therefore, a single transcription error can result in many flawed proteins, whereas a translation error will disrupt only a single protein. Estimates of the rate of transcription errors in Escherichia coli have yielded variable estimates of transcription error rates of 10−4–10−5 per nucleotide, several orders of magnitude higher than the mutation rate . Transcript-error rates are 3 to 4 orders of magnitude higher than the corresponding genetic mutation rates. The general error rates of genomic replication (about 10− are estimated to be approximately 10,000-fold lower than those of protein synthesis (about 10−4), and thus in most instances, mRNA translation is the key process contributing to the inaccuracy of the cellular proteome 4
Gene expression is not only controlled through altering the rate of transcription but also through varying rates of translation and mRNA decay. 3 Alterations in mRNA stability can have dramatic effects on cell physiology and as a consequence the fitness and survival of the organism. Recent evidence suggests that mRNA decay can be regulated in response to environmental cues in order to enable the organism to adapt to its changing surroundings. Bacteria have unique post-transcriptional control mechanisms to enact such adaptive responses through:
1) general mRNA decay,
2) differential mRNA degradation using small non-coding RNAs (sRNAs), and
3) selective mRNA degradation using the tmRNA quality control system.
As with other biomolecules, the great variety of RNA species produced by the bacterial cell requires quality control mechanisms to ensure proper folding and function. In addition, the role of mRNA as a template for protein synthesis adds greater significance to mRNA quality control. The translation of a faulty transcript without adequate quality assurance measures might lead to the accumulation of aberrant protein products that could be detrimental to the cell. Bacterial mRNAs are not post-transcriptionally spliced, nor do they exhibit the 5’-cap structures of their eukaryotic counterparts. As such, post-transcriptional quality control processes of prokaryotic mRNA are distinct from the corresponding processes in eukaryotes. This review focuses on issues related to post-transcriptional processing, targeting, and degradation of bacterial mRNAs including those facilitated by small regulatory RNAs, with special emphasis on tmRNA and trans-translation.
Variation in the stability of transcripts has an important role in the control of protein expression within the cell, as long-lived transcripts are generally subject to more rounds of translation than those with a shorter half-life. Several factors play a role in controlling the lifespan of specific mRNAs by regulating their propensity to be degraded.
Translational decoding of the mRNA codons is constrained by factors during codon-anticodon recognition and often constitutes the rate-limiting step during protein synthesis. Besides the abundance of tRNA species, mRNA translation is regulated by nearly 100 epigenetic tRNA modifications, especially at the wobble position 4 Faithful translation of the mRNA codons into protein is essential for cellular physiology. The fidelity of the translation machinery firstly depends on the specific coupling of amino acids to their cognate tRNA species, which is catalyzed by aminoacyl-tRNA synthetases (aaRSs). Eukaryotic elongation factor 1A (eEF-1A) or prokaryotic EF-Tu delivers the aminoacyl-tRNA to the ribosome A site for elongation of nascent peptide chain after proper codon-anticodon recognition. Thus, aminoacyl-tRNA synthetase (aaRSs) are cardinal in protecting protein synthesis against misacylation ( incorrectly adding an acyl group to a compound).
tRNA synthesis quality control
Hypomodified tRNAs (modified to an abnormally small degree) can be degraded by the RNA degradosome, a multicomponent RNA degradation complex. Thus, the degradosome serves as a previously unrecognized bacterial tRNA quality control system that mediates clearance of hypomodified tRNAs.can be degraded by the RNA degradosome, a multicomponent RNA degradation complex. Thus, the degradosome serves as a bacterial tRNA quality control system that mediates clearance of hypomodified tRNAs. 1 Within the anticodon, the first or “wobble” position is particularly subject to modification, and these modifications are often critical for efficient translation; consequently, mutations that prevent such modification can result in loss of viability. tRNAs’ highly stable structures are thought to contribute to their intracellular stability; in general, tRNAs exhibit a high melting temperature in vitro and a long half-life within the cell. RTD and other mechanisms that degrade hypomodified or mutated mature yeast tRNAs serve as a surveillance system to eliminate tRNA molecules that have incorrect nucleosides or conformations
In order to ensure fidelity, some of the synthetases perform editing functions to reduce the occurrence of errors during protein synthesis. 5
Quality control mechanisms during ribosome maturation
Protein synthesis on ribosomes is carefully quality controlled to ensure the faithful transmission of genetic information from mRNA to protein. Many of these mechanisms rely on communication between distant sites on the ribosomes, and thus on the integrity of the ribosome structure. Furthermore, haploinsufficiency of ribosomal proteins, which increases the chances of forming incompletely assembled ribosomes, can predispose to cancer. Finally, release of inactive ribosomes into the translating pool will lead to their degradation together with the degradation of the bound mRNA. Together, these findings suggest that quality control mechanisms must be in place to survey nascent ribosomes and ensure their functionality. This review gives an account of these mechanisms as currently known. 7
Proof-reading Yeast employs structural proofreading of ribosome functional centres during nuclear steps of assembly
Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome.
We propose that conformational proofreading exerted via Rps20 constitutes a checkpoint permitting assembly factor release and progression of pre-40S maturation only after completion of all earlier maturation steps.
To minimize production of dysfunctional ribosomes, yeast employs structural proofreading of ribosome functional centres during nuclear steps of assembly
Arx1 binding may serve to proofread the proper accommodation of these ribosomal proteins into the tunnel exit
time window for the quality-control machinery to functionally proofread preribosomes.
Error check and repair in the Ribosome: More details come to light
Before an amino acid is added to a growing polypeptide chain, the ribosome folds around the codon–anticodon interaction, and only when the match is correct is this folding completed and the reaction allowed to proceed. Thus, the codon–anticodon interaction is thereby checked twice—once by the initial complementary base-pairing and a second time by the folding of the ribosome, which depends on the correctness of the match. This same principle of induced fit is seen in transcription by RNA polymerase; here, an incoming nucleoside triphosphate initially forms a base pair with the template; at this point the enzyme folds around the base pair (thereby assessing its correctness) and, in doing so, creates the active site of the enzyme. The enzyme then covalently adds the nucleotide to the growing chain. Because their geometry is “wrong,” incorrect base pairs block this induced fit, and they are therefore likely to dissociate before being incorporated into the growing chain. A second principle used to increase the specificity of complementary base-pairing is called kinetic proofreading. We have seen that after the initial codon‒anticodon pairing and conformational change of the ribosome, GTP is hydrolyzed. This creates an irreversible step and starts the clock on a time delay during which the aminoacyl-tRNA moves into the proper position for catalysis. During this delay, those incorrect codon–anticodon pairs that have somehow slipped through the induced-fit scrutiny have a higher likelihood of dissociating than correct pairs. There are two reasons for this: (1) the interaction of the wrong tRNA with the codon is weaker, and (2) the delay is longer for incorrect than correct matches. kinetic proofreading thus increases the specificity of complementary base-pairing above what is possible from simple thermodynamic associations alone. The increase in specificity produced by kinetic proofreading comes at an energetic cost in the form of ATP or GTP hydrolysis. Kinetic proofreading is believed to operate in many biological processes, but its role is understood particularly well for translation.
Accuracy in Translation Requires an Expenditure of Free Energy
Translation by the ribosome is a compromise between the opposing constraints of accuracy and speed. We have seen, for example, that the accuracy of translation (1 mistake per 104 amino acids joined) requires time delays each time a new amino acid is added to a growing polypeptide chain, producing an overall speed of translation of 20 amino acids incorporated per second in bacteria. Mutant bacteria with a specific alteration in the small ribosomal subunit have longer delays and translate mRNA into protein with an accuracy considerably higher than this; however, protein synthesis is so slow in these mutants that the bacteria are barely able to survive.
Question: How could the right speed have been obtained with trial and error, if slow mutants do not survive? Had the speed not to be right right from the beginning?
We have also seen that attaining the observed accuracy of protein synthesis requires the expenditure of a great deal of free energy; this is expected, since, there is a price to be paid for any increase in order in the cell. In most cells, protein synthesis consumes more energy than any other biosynthetic process. At least four high-energy phosphate bonds are split to make each new peptide bond: two are consumed in charging a tRNA molecule with an amino acid, and two more drive steps in the cycle of reactions occurring on the ribosome during protein synthesis itself. In addition, extra energy is consumed each time that an incorrect amino acid linkage is hydrolyzed by a tRNA synthetase and each time that an incorrect tRNA enters the ribosome, triggers GTP hydrolysis, and is rejected. To be effective, any proofreading mechanism must also allow an appreciable fraction of correct interactions to be removed; for this reason, proofreading is even more costly in energy than it might at first seem.
My comment: So we see the same proofreading principles applied in totally distinct protein complexes. Furthermore, proof reading requires energy. This is, therefore, clear evidence of design.
Carnegie Institution for Science Quality control mechanism closes the protein production 'on-ramps' in cells OCTOBER 8, 2020
Quality control mechanism closes the protein production 'on-ramps' in cells
Recent work led by Carnegie's Kamena Kostova revealed a new quality control system in the protein production assembly line with possible implications for understanding neurogenerative disease.
The DNA that comprises the chromosomes housed in each cell's nucleus encodes the recipes for how to make proteins, which are responsible for the majority of the physiological actions that sustain life. Individual recipes are transcribed using messenger RNA, which carries this piece of code to a piece of cellular machinery called the ribosome. The ribosome translates the message into amino acids—the building blocks of proteins.
But sometimes messages get garbled. The resulting incomplete protein products can be toxic to cells. So how do cells clean up in the aftermath of a botched translation?
Some quality assurance mechanisms were already known—including systems that degrade the half-finished protein product and the messenger RNA that led to its creation. But Kostova led a team that identified a new tool in the cell's kit for preventing damage when protein assembly goes awry. Their work was published by Molecular Cell.
Using CRISPR-Cas9-based genetic screening, the researchers discovered a separate, and much needed, device by which the cell prevents that particular faulty message from being translated again. They found two factors, called GIGYF2 and 4EHP, which prevent translation from being initiated on problematic messenger RNA fragments.
"Imagine that the protein assembly process is a highway and the ribosomes are cars traveling on it," Kostova explained. "If there's a bad message producing incomplete protein products, it's like having a stalled car or two on the road, clogging traffic. Think of GIGYF2 and 4EHP as closing the on-ramp, so that there is time to clear everything away and additional cars don't get stalled, exacerbating the problem."
Loss of GIGYF2 has previously been associated with neurodegenerative and neurodevelopmental problems. It is possible that these issues are caused by the buildup of defective proteins that occurs without the ability to prevent translation on faulty messenger RNAs.
Ka-Weng Ieong Two proofreading steps amplify the accuracy of genetic code translation November 11, 2016
We have discovered that two proofreading steps amplify the accuracy of genetic code reading, not one step, as hitherto believed. We have characterized the molecular basis of each one of these steps, paving the way for structural analysis in conjunction with structure-based standard free energy computations. Our work highlights the essential role of elongation factor Tu for accurate genetic code translation in both initial codon selection and proofreading. Our results have implications for the evolution of efficient and accurate genetic code reading through multistep proofreading, which attenuates the otherwise harmful effects of the obligatory tradeoff between efficiency and accuracy in substrate selection by enzymes.
There is a vast network of information flow in a typical cell, and along with that flow there is a vast network of error checking. Damage to DNA sequences is remedied, the transcribing of DNA is checked and corrected, and at the ribosome the translation process is checked and controlled. In fact, recent research has found that the ribosome not only carefully sets up the codon-to-amino-acid translation process for success, but if an error is made the ribosome detects it and takes action after the translation process. When the ribosome detects a translation error it takes action 10,000 times faster than it normally does. "These are not subtle numbers," explained the lead researcher. As one report explains, "the ribosome exerts far tighter quality control than anyone ever suspected." How does the ribosome do it? The ribosome--which creates proteins--consists of RNA and protein molecules. If the ribosome is the machine that builds proteins, then from where did the ribosome's proteins come in the first place? Evolutionists believe that initial versions of the ribosome--the proto-ribosome--had only the RNA molecules and the proteins came later. Perhaps so, but the translation task is not simple, and the ribosome's proteins do not appear simply to be innocent bystanders that evolution, for no particular reason, kludged onto the ribosome. Rather, the proteins are deeply embedded in the ribosome, and appear to be important for both the ribosome's structure construction and conformation. This is probably why RNA-only proto-ribosomes don't seem to work. But this is not all. Even ignoring the problem of obtaining an RNA-only translation machine, the evolutionary hypothesis raises the question: From where did the protein-coding sequences come which it would translate? In other words, even if a long sequence of RNA residues just happened to assemble and fold and function as a proto-ribosome, why would it be selected for if there were no protein-coding sequences lying around? One could add to this a long list of other requirements, such as a ready made pool of amino acids, and of course something for the newly minted protein to do. Of course evolutionists can always speculate. For instance, perhaps a functional RNA molecule just happened to also code for a useful protein. How convenient. Fortunately, in a world where confessions of evolution's heroics are rare, one nobel laureate scientist gave this judicious observation: "How evolution managed to progress from making a random peptide to messenger-directed synthesis, we haven't a clue." And yet evolution is a fact? I think I want my money back.