Re: [ccp4bb] Reverse Translatase
On 09/07/10 22:10, Jacob Keller wrote: In terms of usefulness, I was actually thinking about cells learning how to make new proteins from other cells, Which they do already by exchanging genes or perhaps an immune system could use the info to make the right choice of starting materials. The methods by which the immune system works have been at least partially elucidated, and are available for study. Also, codon bias could be explained as resulting from the nature of the reverse translatase machinery. 1) Or, you could explain it as resulting from the nature of unicorns. You might understand that this would be an easier sell if you could first establish the existence of unicorns. 2) What is it about codon bias which you feel requires such an elaborate explanation? Or an invader could copy the host's membrane proteins to evade detection. They can do that now by 1) Stealing the host's genes 2) Stealing existing peptides and lipids and grafting them to their surface Ah, so many possibilities! And as I said before, considering that it would be so useful, and that the genius of macromolecular design observed in nature is apparently so unlimited, shouldn't it be out there somewhere? Design? I think there are more appropriate descriptions for life as it has been observed. The complexity of life can be explained fairly well by Darwinian evolution, i.e. replication with variation coupled with selection. This works through modification of existing entities. The relatedness of many molecules and the theme of modification of pre-existing parts ought to be apparent to someone who has learned about replication and sources of genetic novelty, and spent any time studying protein structure. The large barriers to the introduction of your reverse translation system have already been pointed out. Come up with a Darwinian sequence of how it could have developed gradually from existing systems and get back to us. For comparisons, Darwinian explanations for the development of ribosomal translation and the genetic code have been proferred. I want it, therefore it should exist doesn't cut it. Nobel prize to the one who finds it... Certainly. NB It should not cross our minds, I don't think, that if it were there, it would have been found. Small RNA phenomena, for example, went undetected for years, despite their commonness and high importance. Now that we have access to many complete genomes, if genes were being swapped by your reverse translatase system rather than horizontal gene transfer, the results should be readily apparent. Cheers, -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Reverse Translatase
Hi Jacob, A couple more things to think about: 1.) How to get a Nobel Prize: Try an experiment if you have the means. Transform/transfect your favorite cell type with exogenous protein sequences. Sequence and see if they ever appear in the genome. Go after it... 2.) I realize that the idea of a reverse translatase doesn't necessarily imply that the sequence ever makes it into the genome, but if this is to be a truly useful biological system, it would need to include a reverse transcription and incorporation into the genome in order to pass the acquired information to daughter cells. Imagine the scenario in which the pathogen evades the immune system by copying host proteins. If they could not pass this information to daughter cells as they divide, those cells would instantly be susceptible to the immune system. There needs to be some selective pressure, an acquired benefit that can be passed to offspring, that would make this system truly biologically useful. Now comes the question of how does this reverse translated/transcribed gene get into the genome. A transposase, sure, but how does this transposase know where to put the gene? It must be the appropriate distance from an appropriate promoter and enhancers, etc. otherwise it will not be expressed properly. And it has to have ribosome binding motifs (which would still be required even without genomic incorporation). Remember, the huge hurdle to much of gene therapy has been controlling where the exogenous gene inserts into the genome. Some incorporate in a highly nonspecific manner, in far too many places and/or interfere with other vital genes as they insert and in doing so cause a number of terrible side-effects like cancers, etc. 3.) Codon bias has already been sufficiently explained by correlating codon usage with the expression/availability of their associated tRNAs. This has been proved experimentally, and I don't have a reference for this, but consider certain commercial competent cell strains for expression in E.coli. Cells such as BL21(DE3)-RILP are so effective in expressing proteins from different organisms from expression vectors, even without codon optimization for bacterial expression, simply because they contain plasmids that also overexpress certain rare tRNAs (for Arg, Ile, Leu, and Pro in the cell line mentioned), thereby increasing their availability and subsequent usage. Keep chasing that Nobel Prize, but have a backup plan...you're clearly very creative. Mike - Original Message - From: Jacob Keller j-kell...@fsm.northwestern.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, September 7, 2010 7:10:02 PM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Reverse Translatase In terms of usefulness, I was actually thinking about cells learning how to make new proteins from other cells, or perhaps an immune system could use the info to make the right choice of starting materials. Also, codon bias could be explained as resulting from the nature of the reverse translatase machinery. Or an invader could copy the host's membrane proteins to evade detection. Ah, so many possibilities! And as I said before, considering that it would be so useful, and that the genius of macromolecular design observed in nature is apparently so unlimited, shouldn't it be out there somewhere? Nobel prize to the one who finds it... Jacob NB It should not cross our minds, I don't think, that if it were there, it would have been found. Small RNA phenomena, for example, went undetected for years, despite their commonness and high importance. - Original Message - From: Artem Evdokimov ar...@xtals.org To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, September 07, 2010 8:29 PM Subject: Re: [ccp4bb] Reverse Translatase Regardless of whether a system like this exists in Nature or not - it's fun to imagine! On a microscopic scale one could propose a hypothetical mechanism by which a completely unfolded polypeptide chain could be fed into a gated (or state-locked) peptidase that may break the chain down in a co-ordinated stepwise fashion; releasing individual aa's into some sort of a nanoscale channel. The released aa's would then be sequentially coupled to something resembling tRNA - with pre-formed trinucleotides attached on the other end. Coupling would then presumably permit the triplets to ligate to one another sequentially - the resulting ssDNA or ssRNA would then have to be converted into a stable ds-form via the usual means, or otherwise protected in one of the usual ways. Codon space could be expanded by pre-loading carrier molecules with more than one type of triplet per carrier (biased towards whatever codon frequencies are prominent in the organism of choice) although this in no way resolves the random nature of the actual codon use within the resulting nucleotide sequence. The issue of amino acid coupling selectivity is pretty hairy - the best I could think of on a short notice is to have
Re: [ccp4bb] Reverse Translatase
Hi Jacob and Mike, An additional thought exercise would be to consider if your exogenously added protein is itself heritable. Essentially trying Mike's idea 1, but instead of expecting the protein to get into the genome and in that way have it's information stored, look to see if the protein itself is passed down, because if a reverse translatase existed the information may be encoded in the protein. -bob On Wed, Sep 8, 2010 at 5:31 PM, Michael Thompson mi...@chem.ucla.edu wrote: Hi Jacob, A couple more things to think about: 1.) How to get a Nobel Prize: Try an experiment if you have the means. Transform/transfect your favorite cell type with exogenous protein sequences. Sequence and see if they ever appear in the genome. Go after it... 2.) I realize that the idea of a reverse translatase doesn't necessarily imply that the sequence ever makes it into the genome, but if this is to be a truly useful biological system, it would need to include a reverse transcription and incorporation into the genome in order to pass the acquired information to daughter cells. Imagine the scenario in which the pathogen evades the immune system by copying host proteins. If they could not pass this information to daughter cells as they divide, those cells would instantly be susceptible to the immune system. There needs to be some selective pressure, an acquired benefit that can be passed to offspring, that would make this system truly biologically useful. Now comes the question of how does this reverse translated/transcribed gene get into the genome. A transposase, sure, but how does this transposase know where to put the gene? It must be the appropriate distance from an appropriate promoter and enhancers, etc. otherwise it will not be expressed properly. And it has to have ribosome binding motifs (which would still be required even without genomic incorporation). Remember, the huge hurdle to much of gene therapy has been controlling where the exogenous gene inserts into the genome. Some incorporate in a highly nonspecific manner, in far too many places and/or interfere with other vital genes as they insert and in doing so cause a number of terrible side-effects like cancers, etc. 3.) Codon bias has already been sufficiently explained by correlating codon usage with the expression/availability of their associated tRNAs. This has been proved experimentally, and I don't have a reference for this, but consider certain commercial competent cell strains for expression in E.coli. Cells such as BL21(DE3)-RILP are so effective in expressing proteins from different organisms from expression vectors, even without codon optimization for bacterial expression, simply because they contain plasmids that also overexpress certain rare tRNAs (for Arg, Ile, Leu, and Pro in the cell line mentioned), thereby increasing their availability and subsequent usage. Keep chasing that Nobel Prize, but have a backup plan...you're clearly very creative. Mike - Original Message - From: Jacob Keller j-kell...@fsm.northwestern.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, September 7, 2010 7:10:02 PM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Reverse Translatase In terms of usefulness, I was actually thinking about cells learning how to make new proteins from other cells, or perhaps an immune system could use the info to make the right choice of starting materials. Also, codon bias could be explained as resulting from the nature of the reverse translatase machinery. Or an invader could copy the host's membrane proteins to evade detection. Ah, so many possibilities! And as I said before, considering that it would be so useful, and that the genius of macromolecular design observed in nature is apparently so unlimited, shouldn't it be out there somewhere? Nobel prize to the one who finds it... Jacob NB It should not cross our minds, I don't think, that if it were there, it would have been found. Small RNA phenomena, for example, went undetected for years, despite their commonness and high importance. - Original Message - From: Artem Evdokimov ar...@xtals.org To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, September 07, 2010 8:29 PM Subject: Re: [ccp4bb] Reverse Translatase Regardless of whether a system like this exists in Nature or not - it's fun to imagine! On a microscopic scale one could propose a hypothetical mechanism by which a completely unfolded polypeptide chain could be fed into a gated (or state-locked) peptidase that may break the chain down in a co-ordinated stepwise fashion; releasing individual aa's into some sort of a nanoscale channel. The released aa's would then be sequentially coupled to something resembling tRNA - with pre-formed trinucleotides attached on the other end. Coupling would then presumably permit the triplets to ligate to one another sequentially - the resulting ssDNA or ssRNA would then have
Re: [ccp4bb] Reverse Translatase
David is absolutely right. There is no design, Jacob, we just instinctively look for it everywhere because seeking purpose instead of understanding mechanism conveys advantage to our species. Your rationale is flawed - just because it is imaginable (with caveats) does not mean that it must exist on this particular planet. Complementary, not every feature observed has functional significance (in part because biomacromolecules are structurally redundant). On Wed, 2010-09-08 at 09:04 -0400, David Schuller wrote: Ah, so many possibilities! And as I said before, considering that it would be so useful, and that the genius of macromolecular design observed in nature is apparently so unlimited, shouldn't it be out there somewhere? Design? I think there are more appropriate descriptions for life as it has been observed. The complexity of life can be explained fairly well by Darwinian evolution, i.e. replication with variation coupled with selection. This works through modification of existing entities. The relatedness of many molecules and the theme of modification of pre-existing parts ought to be apparent to someone who has learned about replication and sources of genetic novelty, and spent any time studying protein structure.
Re: [ccp4bb] Reverse Translatase
A large amount of hypotheticals can be found in Plasmodium :-) Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 8, 2010, at 9:29 PM, Ed Pozharski wrote: David is absolutely right. There is no design, Jacob, we just instinctively look for it everywhere because seeking purpose instead of understanding mechanism conveys advantage to our species. Your rationale is flawed - just because it is imaginable (with caveats) does not mean that it must exist on this particular planet. Complementary, not every feature observed has functional significance (in part because biomacromolecules are structurally redundant). On Wed, 2010-09-08 at 09:04 -0400, David Schuller wrote: Ah, so many possibilities! And as I said before, considering that it would be so useful, and that the genius of macromolecular design observed in nature is apparently so unlimited, shouldn't it be out there somewhere? Design? I think there are more appropriate descriptions for life as it has been observed. The complexity of life can be explained fairly well by Darwinian evolution, i.e. replication with variation coupled with selection. This works through modification of existing entities. The relatedness of many molecules and the theme of modification of pre-existing parts ought to be apparent to someone who has learned about replication and sources of genetic novelty, and spent any time studying protein structure.
Re: [ccp4bb] Reverse Translatase
So, let's say instead these adaptors recognize 2 amino acids at a time (still probably not robust enough). Then, one would need 2^20 adaptors, already a far greater number of gene products than that present in any genome than I know of... Surely only 20^2, which is 400? A lot, but managable. James -- Dr. James W. Murray David Phillips Research Fellow Division on Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Greg Alushin [galus...@berkeley.edu] Sent: Tuesday, September 07, 2010 3:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Reverse Translatase Hi Jacob- What an intriguing proposition. I can think of multiple reasons why such a system would not exist, but there is a mechanistic one which is most fundamental, having to do with the nature of the genetic code. Say that there is a cellular machine which would unfold a protein and expose it to some sort of reading system (already a hard problem). There is now the issue of transforming the amino acid information into nucleic acid information. For simplicity let's assume that this system only uses one codon per amino acid, bypassing the degeneracy problem. How would the cell then read off the amino acid sequence? It seems the simplest solution would be analogous to translation, i.e. having adaptor molecules analogues to tRNAs which would guide an enzyme that synthesized the nucleic acid. Otherwise, one would have to invoke the idea of a single enzyme recognizing every amino acid, which seems impossible to me. As we know, the problem of protein-protein recognition is relatively complex. At a minimum, one would need 20 adaptor proteins to recognize the 20 canonical amino acids: however, it seems unlikely that recognition of a single amino acid would be robust enough to select for the correct adaptor molecule. So, let's say instead these adaptors recognize 2 amino acids at a time (still probably not robust enough). Then, one would need 2^20 adaptors, already a far greater number of gene products than that present in any genome than I know of... It might be tempting to draw an analogy between this system and the immune system, where an incredible diversity is generated from a small number of genes. However, diverse immune proteins all take the same input sequence (say antigen recognition) and lead to a single response, whereas this system has a 1 to 1 correspondence between inputs (protein sequence) and outputs (nucleic acid sequences), and thus there is no way that a randomization system could generate the required diversity. Cheers, -Greg Alushin Nogales lab UC Berkeley On Sep 6, 2010, at 7:12 PM, Michael Thompson wrote: Jacob, The idea is enticing, but don't forget that there are multiple degenerate codons for a given amino acid. Once the protein is synthesized, the specific codon information is lost. I think that's a fundamental problem. Keep the ideas coming, Mike Thompson - Original Message - From: Jacob Keller j-kell...@fsm.northwestern.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 6, 2010 6:36:14 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Reverse Translatase Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. Best Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Reverse Translatase
Whoops, sorry of course that is right. But 3 amino acids would be 8000, etc. -Greg On Sep 7, 2010, at 12:46 AM, Murray, James W wrote: So, let's say instead these adaptors recognize 2 amino acids at a time (still probably not robust enough). Then, one would need 2^20 adaptors, already a far greater number of gene products than that present in any genome than I know of... Surely only 20^2, which is 400? A lot, but managable. James -- Dr. James W. Murray David Phillips Research Fellow Division on Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Greg Alushin [galus...@berkeley.edu] Sent: Tuesday, September 07, 2010 3:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Reverse Translatase Hi Jacob- What an intriguing proposition. I can think of multiple reasons why such a system would not exist, but there is a mechanistic one which is most fundamental, having to do with the nature of the genetic code. Say that there is a cellular machine which would unfold a protein and expose it to some sort of reading system (already a hard problem). There is now the issue of transforming the amino acid information into nucleic acid information. For simplicity let's assume that this system only uses one codon per amino acid, bypassing the degeneracy problem. How would the cell then read off the amino acid sequence? It seems the simplest solution would be analogous to translation, i.e. having adaptor molecules analogues to tRNAs which would guide an enzyme that synthesized the nucleic acid. Otherwise, one would have to invoke the idea of a single enzyme recognizing every amino acid, which seems impossible to me. As we know, the problem of protein-protein recognition is relatively complex. At a minimum, one would need 20 adaptor proteins to recognize the 20 canonical amino acids: however, it seems unlikely that recognition of a single amino acid would be robust enough to select for the correct adaptor molecule. So, let's say instead these adaptors recognize 2 amino acids at a time (still probably not robust enough). Then, one would need 2^20 adaptors, already a far greater number of gene products than that present in any genome than I know of... It might be tempting to draw an analogy between this system and the immune system, where an incredible diversity is generated from a small number of genes. However, diverse immune proteins all take the same input sequence (say antigen recognition) and lead to a single response, whereas this system has a 1 to 1 correspondence between inputs (protein sequence) and outputs (nucleic acid sequences), and thus there is no way that a randomization system could generate the required diversity. Cheers, -Greg Alushin Nogales lab UC Berkeley On Sep 6, 2010, at 7:12 PM, Michael Thompson wrote: Jacob, The idea is enticing, but don't forget that there are multiple degenerate codons for a given amino acid. Once the protein is synthesized, the specific codon information is lost. I think that's a fundamental problem. Keep the ideas coming, Mike Thompson - Original Message - From: Jacob Keller j-kell...@fsm.northwestern.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 6, 2010 6:36:14 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Reverse Translatase Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. Best Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Reverse Translatase
On 09/06/10 21:36, Jacob Keller wrote: Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. See: The RNA/Protein Symmetry Hypothesis: Experimental Support for Reverse Translation of Primitive Proteins Masayuki Nahimoto, J. Theor. Biol. (2001) 209, pp 181-187. In which Nahimoto proposes such a system, and additionally proposes that it actually existed early in the development of life on this planet. Reasons why it could not exist - No. Reasons why it would be very difficult - yes. And plenty of reasons why Nahimoto is probably wrong about it having actually existed: There is absolutely no evidence presented that such a system was ever in operation in the history of life on this planet. Current theories such as the RNA World are much more likely explanations for how life as we currently know it may have developed from a pre-biotic state. DNA replication, DNA=RNA transcription, and RNA=Protein translation all depend on nucleic acid base pairing for part of their specificity. It truly is the secret of life. And it would not be especially helpful in Protein=RNA reverse translation. Forward translation takes place in the ribosome, but extra specificity is smuggled in via a large set of tRNAs and tRNA charging enzymes, in reactions which took place beforehand, which are then made use of through the base-pairing codon:anti-codon recognition. Reverse translation would most definitely not be running forward translation in reverse; the specificity cannot be handled ahead of time, it needs to be available at the site of reverse translation itself when each successive peptide residue is presented. Progressivity: If different recognition sites are swapped in, this has to be done while keeping place in both the protein chain and in the growing nucleotide chain. Possibly the protein chain might be cleaved during the process. The chemistry and geometry of peptide residues is far more variable than that of nucleotide residues. The genetic code of reverse translation would be completely independent of that in forward translation. For the two to have matched up (in the proposed naturally occurring RT system) would have been extremely fortuitous, imposing a strong barrier to the introduction of such a system. Difficulty in dealing with post-translational modifications disulfides, cyclical peptides, acetylation, phosphorylation, etc. A peptide sequencer coupled with a nucleotide synthesizer accomplishes somewhat the same thing, but on a macroscopic scale. This is an impediment to the motivation for constructing a reverse translatase enzymatic system. Cheers, -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Reverse Translatase
Regardless of whether a system like this exists in Nature or not - it's fun to imagine! On a microscopic scale one could propose a hypothetical mechanism by which a completely unfolded polypeptide chain could be fed into a gated (or state-locked) peptidase that may break the chain down in a co-ordinated stepwise fashion; releasing individual aa's into some sort of a nanoscale channel. The released aa's would then be sequentially coupled to something resembling tRNA - with pre-formed trinucleotides attached on the other end. Coupling would then presumably permit the triplets to ligate to one another sequentially - the resulting ssDNA or ssRNA would then have to be converted into a stable ds-form via the usual means, or otherwise protected in one of the usual ways. Codon space could be expanded by pre-loading carrier molecules with more than one type of triplet per carrier (biased towards whatever codon frequencies are prominent in the organism of choice) although this in no way resolves the random nature of the actual codon use within the resulting nucleotide sequence. The issue of amino acid coupling selectivity is pretty hairy - the best I could think of on a short notice is to have the receptor sites for individual aa's arranged in order of dropping selectivity -- however there is still the matter of shape/property similarities throwing wrenches into the works. An alternative would be a series of binary gates working on an exclusion principle. As to practicality of this kind of stuff - I am not sure; I can imagine an application similar to nano-scale multiparallel pyrosequencing: an unknown protein would be broken down into peptides via nonselective protease of some sort and then relatively short individual peptides are 'sequenced' in parallel, producing short DNA sequences that would later be complemented to dsDNA and allowed to cross-anneal and self-assemble via overlaps, similar to gapped gene assembly from short synthetic fragments (that first protease better be *really* non-specific!). At the end one could sequence the resulting long DNA to see what the original protein was like. A. On Tue, Sep 7, 2010 at 8:35 AM, David Schuller dj...@cornell.edu wrote: On 09/06/10 21:36, Jacob Keller wrote: Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. See: The RNA/Protein Symmetry Hypothesis: Experimental Support for Reverse Translation of Primitive Proteins Masayuki Nahimoto, J. Theor. Biol. (2001) 209, pp 181-187. In which Nahimoto proposes such a system, and additionally proposes that it actually existed early in the development of life on this planet. Reasons why it could not exist - No. Reasons why it would be very difficult - yes. And plenty of reasons why Nahimoto is probably wrong about it having actually existed: There is absolutely no evidence presented that such a system was ever in operation in the history of life on this planet. Current theories such as the RNA World are much more likely explanations for how life as we currently know it may have developed from a pre-biotic state. DNA replication, DNA=RNA transcription, and RNA=Protein translation all depend on nucleic acid base pairing for part of their specificity. It truly is the secret of life. And it would not be especially helpful in Protein=RNA reverse translation. Forward translation takes place in the ribosome, but extra specificity is smuggled in via a large set of tRNAs and tRNA charging enzymes, in reactions which took place beforehand, which are then made use of through the base-pairing codon:anti-codon recognition. Reverse translation would most definitely not be running forward translation in reverse; the specificity cannot be handled ahead of time, it needs to be available at the site of reverse translation itself when each successive peptide residue is presented. Progressivity: If different recognition sites are swapped in, this has to be done while keeping place in both the protein chain and in the growing nucleotide chain. Possibly the protein chain might be cleaved during the process. The chemistry and geometry of peptide residues is far more variable than that of nucleotide residues. The genetic code of reverse translation would be completely independent of that in forward translation. For the two to have matched up (in the proposed naturally occurring RT system) would have been extremely fortuitous, imposing a strong barrier to the introduction of such a system. Difficulty in dealing with post-translational modifications disulfides, cyclical peptides, acetylation, phosphorylation, etc. A peptide sequencer coupled with a nucleotide synthesizer accomplishes
Re: [ccp4bb] Reverse Translatase
In terms of usefulness, I was actually thinking about cells learning how to make new proteins from other cells, or perhaps an immune system could use the info to make the right choice of starting materials. Also, codon bias could be explained as resulting from the nature of the reverse translatase machinery. Or an invader could copy the host's membrane proteins to evade detection. Ah, so many possibilities! And as I said before, considering that it would be so useful, and that the genius of macromolecular design observed in nature is apparently so unlimited, shouldn't it be out there somewhere? Nobel prize to the one who finds it... Jacob NB It should not cross our minds, I don't think, that if it were there, it would have been found. Small RNA phenomena, for example, went undetected for years, despite their commonness and high importance. - Original Message - From: Artem Evdokimov ar...@xtals.org To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, September 07, 2010 8:29 PM Subject: Re: [ccp4bb] Reverse Translatase Regardless of whether a system like this exists in Nature or not - it's fun to imagine! On a microscopic scale one could propose a hypothetical mechanism by which a completely unfolded polypeptide chain could be fed into a gated (or state-locked) peptidase that may break the chain down in a co-ordinated stepwise fashion; releasing individual aa's into some sort of a nanoscale channel. The released aa's would then be sequentially coupled to something resembling tRNA - with pre-formed trinucleotides attached on the other end. Coupling would then presumably permit the triplets to ligate to one another sequentially - the resulting ssDNA or ssRNA would then have to be converted into a stable ds-form via the usual means, or otherwise protected in one of the usual ways. Codon space could be expanded by pre-loading carrier molecules with more than one type of triplet per carrier (biased towards whatever codon frequencies are prominent in the organism of choice) although this in no way resolves the random nature of the actual codon use within the resulting nucleotide sequence. The issue of amino acid coupling selectivity is pretty hairy - the best I could think of on a short notice is to have the receptor sites for individual aa's arranged in order of dropping selectivity -- however there is still the matter of shape/property similarities throwing wrenches into the works. An alternative would be a series of binary gates working on an exclusion principle. As to practicality of this kind of stuff - I am not sure; I can imagine an application similar to nano-scale multiparallel pyrosequencing: an unknown protein would be broken down into peptides via nonselective protease of some sort and then relatively short individual peptides are 'sequenced' in parallel, producing short DNA sequences that would later be complemented to dsDNA and allowed to cross-anneal and self-assemble via overlaps, similar to gapped gene assembly from short synthetic fragments (that first protease better be *really* non-specific!). At the end one could sequence the resulting long DNA to see what the original protein was like. A. On Tue, Sep 7, 2010 at 8:35 AM, David Schuller dj...@cornell.edu wrote: On 09/06/10 21:36, Jacob Keller wrote: Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. See: The RNA/Protein Symmetry Hypothesis: Experimental Support for Reverse Translation of Primitive Proteins Masayuki Nahimoto, J. Theor. Biol. (2001) 209, pp 181-187. In which Nahimoto proposes such a system, and additionally proposes that it actually existed early in the development of life on this planet. Reasons why it could not exist - No. Reasons why it would be very difficult - yes. And plenty of reasons why Nahimoto is probably wrong about it having actually existed: There is absolutely no evidence presented that such a system was ever in operation in the history of life on this planet. Current theories such as the RNA World are much more likely explanations for how life as we currently know it may have developed from a pre-biotic state. DNA replication, DNA=RNA transcription, and RNA=Protein translation all depend on nucleic acid base pairing for part of their specificity. It truly is the secret of life. And it would not be especially helpful in Protein=RNA reverse translation. Forward translation takes place in the ribosome, but extra specificity is smuggled in via a large set of tRNAs and tRNA charging enzymes, in reactions which took place beforehand, which are then made use of through the base-pairing codon:anti-codon recognition. Reverse translation would most definitely
Re: [ccp4bb] Reverse Translatase
Doesn't natural selection act like a Reverse Translatase? Which is quite an elegant implementation of the idea... On Sep 7, 2010, at 6:29 PM, Artem Evdokimov wrote: Regardless of whether a system like this exists in Nature or not - it's fun to imagine! On a microscopic scale one could propose a hypothetical mechanism by which a completely unfolded polypeptide chain could be fed into a gated (or state-locked) peptidase that may break the chain down in a co-ordinated stepwise fashion; releasing individual aa's into some sort of a nanoscale channel. The released aa's would then be sequentially coupled to something resembling tRNA - with pre-formed trinucleotides attached on the other end. Coupling would then presumably permit the triplets to ligate to one another sequentially - the resulting ssDNA or ssRNA would then have to be converted into a stable ds-form via the usual means, or otherwise protected in one of the usual ways. Codon space could be expanded by pre-loading carrier molecules with more than one type of triplet per carrier (biased towards whatever codon frequencies are prominent in the organism of choice) although this in no way resolves the random nature of the actual codon use within the resulting nucleotide sequence. The issue of amino acid coupling selectivity is pretty hairy - the best I could think of on a short notice is to have the receptor sites for individual aa's arranged in order of dropping selectivity -- however there is still the matter of shape/property similarities throwing wrenches into the works. An alternative would be a series of binary gates working on an exclusion principle. As to practicality of this kind of stuff - I am not sure; I can imagine an application similar to nano-scale multiparallel pyrosequencing: an unknown protein would be broken down into peptides via nonselective protease of some sort and then relatively short individual peptides are 'sequenced' in parallel, producing short DNA sequences that would later be complemented to dsDNA and allowed to cross-anneal and self-assemble via overlaps, similar to gapped gene assembly from short synthetic fragments (that first protease better be *really* non-specific!). At the end one could sequence the resulting long DNA to see what the original protein was like. A. On Tue, Sep 7, 2010 at 8:35 AM, David Schuller dj...@cornell.edu wrote: On 09/06/10 21:36, Jacob Keller wrote: Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. See: The RNA/Protein Symmetry Hypothesis: Experimental Support for Reverse Translation of Primitive Proteins Masayuki Nahimoto, J. Theor. Biol. (2001) 209, pp 181-187. In which Nahimoto proposes such a system, and additionally proposes that it actually existed early in the development of life on this planet. Reasons why it could not exist - No. Reasons why it would be very difficult - yes. And plenty of reasons why Nahimoto is probably wrong about it having actually existed: There is absolutely no evidence presented that such a system was ever in operation in the history of life on this planet. Current theories such as the RNA World are much more likely explanations for how life as we currently know it may have developed from a pre- biotic state. DNA replication, DNA=RNA transcription, and RNA=Protein translation all depend on nucleic acid base pairing for part of their specificity. It truly is the secret of life. And it would not be especially helpful in Protein=RNA reverse translation. Forward translation takes place in the ribosome, but extra specificity is smuggled in via a large set of tRNAs and tRNA charging enzymes, in reactions which took place beforehand, which are then made use of through the base-pairing codon:anti-codon recognition. Reverse translation would most definitely not be running forward translation in reverse; the specificity cannot be handled ahead of time, it needs to be available at the site of reverse translation itself when each successive peptide residue is presented. Progressivity: If different recognition sites are swapped in, this has to be done while keeping place in both the protein chain and in the growing nucleotide chain. Possibly the protein chain might be cleaved during the process. The chemistry and geometry of peptide residues is far more variable than that of nucleotide residues. The genetic code of reverse translation would be completely independent of that in forward translation. For the two to have matched up (in the proposed naturally occurring RT system) would have been extremely fortuitous, imposing a strong barrier to the introduction of such a system. Difficulty
Re: [ccp4bb] Reverse Translatase
I shell correct myself. The Darwin evolution of species is not sufficient to perform all functions of the reverse translatase. The Nature also uses viruses in order to translate proteins from different species. The other forms of reverse translation were probably not needed before the introduction of the immune system. Alex On Sep 7, 2010, at 7:26 PM, aaleshin wrote: Doesn't natural selection act like a Reverse Translatase? Which is quite an elegant implementation of the idea... On Sep 7, 2010, at 6:29 PM, Artem Evdokimov wrote: Regardless of whether a system like this exists in Nature or not - it's fun to imagine! On a microscopic scale one could propose a hypothetical mechanism by which a completely unfolded polypeptide chain could be fed into a gated (or state-locked) peptidase that may break the chain down in a co-ordinated stepwise fashion; releasing individual aa's into some sort of a nanoscale channel. The released aa's would then be sequentially coupled to something resembling tRNA - with pre-formed trinucleotides attached on the other end. Coupling would then presumably permit the triplets to ligate to one another sequentially - the resulting ssDNA or ssRNA would then have to be converted into a stable ds-form via the usual means, or otherwise protected in one of the usual ways. Codon space could be expanded by pre-loading carrier molecules with more than one type of triplet per carrier (biased towards whatever codon frequencies are prominent in the organism of choice) although this in no way resolves the random nature of the actual codon use within the resulting nucleotide sequence. The issue of amino acid coupling selectivity is pretty hairy - the best I could think of on a short notice is to have the receptor sites for individual aa's arranged in order of dropping selectivity -- however there is still the matter of shape/property similarities throwing wrenches into the works. An alternative would be a series of binary gates working on an exclusion principle. As to practicality of this kind of stuff - I am not sure; I can imagine an application similar to nano-scale multiparallel pyrosequencing: an unknown protein would be broken down into peptides via nonselective protease of some sort and then relatively short individual peptides are 'sequenced' in parallel, producing short DNA sequences that would later be complemented to dsDNA and allowed to cross-anneal and self-assemble via overlaps, similar to gapped gene assembly from short synthetic fragments (that first protease better be *really* non-specific!). At the end one could sequence the resulting long DNA to see what the original protein was like. A. On Tue, Sep 7, 2010 at 8:35 AM, David Schuller dj...@cornell.edu wrote: On 09/06/10 21:36, Jacob Keller wrote: Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. See: The RNA/Protein Symmetry Hypothesis: Experimental Support for Reverse Translation of Primitive Proteins Masayuki Nahimoto, J. Theor. Biol. (2001) 209, pp 181-187. In which Nahimoto proposes such a system, and additionally proposes that it actually existed early in the development of life on this planet. Reasons why it could not exist - No. Reasons why it would be very difficult - yes. And plenty of reasons why Nahimoto is probably wrong about it having actually existed: There is absolutely no evidence presented that such a system was ever in operation in the history of life on this planet. Current theories such as the RNA World are much more likely explanations for how life as we currently know it may have developed from a pre- biotic state. DNA replication, DNA=RNA transcription, and RNA=Protein translation all depend on nucleic acid base pairing for part of their specificity. It truly is the secret of life. And it would not be especially helpful in Protein=RNA reverse translation. Forward translation takes place in the ribosome, but extra specificity is smuggled in via a large set of tRNAs and tRNA charging enzymes, in reactions which took place beforehand, which are then made use of through the base-pairing codon:anti-codon recognition. Reverse translation would most definitely not be running forward translation in reverse; the specificity cannot be handled ahead of time, it needs to be available at the site of reverse translation itself when each successive peptide residue is presented. Progressivity: If different recognition sites are swapped in, this has to be done while keeping place in both the protein chain and in the growing nucleotide chain. Possibly the protein chain might be cleaved during the process. The chemistry and geometry of peptide residues is far more variable
Re: [ccp4bb] Reverse Translatase
Jacob, The idea is enticing, but don't forget that there are multiple degenerate codons for a given amino acid. Once the protein is synthesized, the specific codon information is lost. I think that's a fundamental problem. Keep the ideas coming, Mike Thompson - Original Message - From: Jacob Keller j-kell...@fsm.northwestern.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 6, 2010 6:36:14 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Reverse Translatase Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. Best Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Reverse Translatase
Hi Jacob- What an intriguing proposition. I can think of multiple reasons why such a system would not exist, but there is a mechanistic one which is most fundamental, having to do with the nature of the genetic code. Say that there is a cellular machine which would unfold a protein and expose it to some sort of reading system (already a hard problem). There is now the issue of transforming the amino acid information into nucleic acid information. For simplicity let's assume that this system only uses one codon per amino acid, bypassing the degeneracy problem. How would the cell then read off the amino acid sequence? It seems the simplest solution would be analogous to translation, i.e. having adaptor molecules analogues to tRNAs which would guide an enzyme that synthesized the nucleic acid. Otherwise, one would have to invoke the idea of a single enzyme recognizing every amino acid, which seems impossible to me. As we know, the problem of protein-protein recognition is relatively complex. At a minimum, one would need 20 adaptor proteins to recognize the 20 canonical amino acids: however, it seems unlikely that recognition of a single amino acid would be robust enough to select for the correct adaptor molecule. So, let's say instead these adaptors recognize 2 amino acids at a time (still probably not robust enough). Then, one would need 2^20 adaptors, already a far greater number of gene products than that present in any genome than I know of... It might be tempting to draw an analogy between this system and the immune system, where an incredible diversity is generated from a small number of genes. However, diverse immune proteins all take the same input sequence (say antigen recognition) and lead to a single response, whereas this system has a 1 to 1 correspondence between inputs (protein sequence) and outputs (nucleic acid sequences), and thus there is no way that a randomization system could generate the required diversity. Cheers, -Greg Alushin Nogales lab UC Berkeley On Sep 6, 2010, at 7:12 PM, Michael Thompson wrote: Jacob, The idea is enticing, but don't forget that there are multiple degenerate codons for a given amino acid. Once the protein is synthesized, the specific codon information is lost. I think that's a fundamental problem. Keep the ideas coming, Mike Thompson - Original Message - From: Jacob Keller j-kell...@fsm.northwestern.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 6, 2010 6:36:14 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Reverse Translatase Dear Crystallographers, does anyone know of any conceptual reason why a reverse translatase enzyme (protein--nucleic acid) could not exist? I can think of so many things for which such an enzyme would be helpful, both to cells and to scientists...! Unless there is something I am missing, it would seem to me conceptually almost impossible that it *not* exist. Best Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
