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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 <[log in to unmask]> 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" <[log in to unmask]> > To: [log in to unmask] > 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" <[log in to unmask]> > To: <[log in to unmask]> > 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 <[log in to unmask]> 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 >> 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 >> [log in to unmask] >> > > > ******************************************* > 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: [log in to unmask] > ******************************************* > > -- > Michael C. Thompson > > Graduate Student > > Biochemistry & Molecular Biology Division > > Department of Chemistry & Biochemistry > > University of California, Los Angeles > > [log in to unmask] >