Deamination Gradients Research Articles

Deamination gradients within codons after 12 position swap predict amino acid hydrophobicity and parallel β-sheet conformational preference

Deaminations C->T and A->G are frequent mutations producing nucleotide content gradients across genomes proportional to singlestrandedness during replication/transcription. Hence, within single codons, deamination risks increase from first to third codon positions, while second codon positions are functionally most crucial. Here genetic codes are analyzed assuming that after anticodons protected codons from deaminations, first and second codon positions swapped (N2N1N3->N1N2N3), with lowest deamination risks for N2 in presumed primitive N2N1N3 codons. N2N1N3, not standard N1N2N3, codon structure minimizes deaminations inversely proportionally to cognate amino acid hydrophobicity and parallel betasheet conformational preference. For N1N2N3, deamination minimization increases with genetic code integration order of cognate amino acids: during the presumed N2N1N3->N1N2N3 codon structure transition, protein synthesis combined direct codon-amino acid interactions for late amino acids and tRNA-based translation for early amino acids. Hence N2N1N3 codons would correspond to tRNA-free translation by spontaneous codon-amino acid affinities, and tRNA-mediated translation presumably caused N2N1N3->N1N2N3 swaps. Results show that rational, not arbitrary rules link codon and amino acid structures. Some analyses detect mitochondrial RNAs and peptides in public data corresponding to systematic position swaps, suggesting occasional swapping polymerase activity.

The primordial tRNA acceptor stem code from theoretical minimal RNA ring clusters

BackgroundTheoretical minimal RNA rings code by design over the shortest length once for each of the 20 amino acids, a start and a stop codon, and form stem-loop hairpins. This defines at most 25 RNA rings of 22 nucleotides. As a group, RNA rings mimick numerous prebiotic and early life biomolecular properties: tRNAs, deamination gradients and replication origins, emergence of codon preferences for the natural circular code, and contents of early protein coding genes. These properties result from the RNA ring’s in silico design, based mainly on coding nonredundancy among overlapping translation frames, as the genetic code’s codon-amino acid assignments determine. RNA rings resemble ancestral tRNAs, defining RNA ring anticodons and corresponding cognate amino acids. Surprisingly, all examined RNA ring properties coevolve with genetic code integration ranks of RNA ring cognates, as if RNA rings mimick prebiotic and early life evolution.MethodsDistances between RNA rings were calculated using different evolutionary models. Associations between these distances and genetic code evolutionary hypotheses detect evolutionary models best describing RNA ring diversification.ResultsHere pseudo-phylogenetic analyses of RNA rings produce clusters corresponding to the primordial code in tRNA acceptor stems, more so when substitution matrices from neutrally evolving pseudogenes are used rather than from functional protein coding genes reflecting selection for conserving amino acid properties.ConclusionsResults indicate RNA rings with recent cognates evolved from those with early cognates. Hence RNA rings, as designed by the genetic code’s structure, simulate tRNA stem evolution and prebiotic history along neutral chemistry-driven mutation regimes.

Theoretical minimal RNA rings designed according to coding constraints mimic deamination gradients

Deaminations (A->G, C->T) increase with DNA singlestrandedness during replication, presumably creating spontaneous genomic mutational and nucleotide frequency gradients. Alternatively, genes are positioned to avoid deaminations. Deamination gradients affect directly mitogene third codon positions; conserved vertebrate mitochondrial tRNA and protein coding gene arrangements minimize deaminations in anticodons, and first and second codon positions in mitogenes. Here we describe deamination gradients across theoretical minimal RNA rings, 22 nucleotide-long RNAs designed to simulate prebiotic RNAs. These RNA rings code for a start/stop codon and a single codon for each amino acid, and form stem-loop hairpins slowing degradation. They resemble consensus tRNAs, defining potential anticodons and cognate amino acids. Theoretical minimal RNA rings are not designed to include deamination gradients, yet deamination gradients occur in RNA rings. tRNA homology produces stronger evidence for deamination gradients than RNA ring homology defined by coding properties. Deamination gradients start at predicted RNA ring anticodons, corresponding to known homologies between mitochondrial tRNAs and replication origins, and between bacterial tRNA synthetases and mitochondrial DNA polymerase gamma. Deamination gradients are strongest for RNA rings with predicted anticodons matching cognate amino acids that integrated early the genetic code. Presumably protections against deaminations evolved while amino acids integrated the genetic code. Results confirm tRNA-RNA ring homologies. Coding constraints defining RNA rings presumably produce deamination gradients starting at predicted anticodons. Hence, the universal genetic code determines nucleotide deamination gradients in theoretical minimal RNA rings, suggesting adaptation to prevent consequences of deamination mutations. Results also indicate that the genetic code's structure determined evolution of tRNAs, their cognates, tRNA synthetases, and polymerases.

Triplex DNA:RNA, 3′-to-5′ Inverted RNA and Protein Coding in Mitochondrial Genomes

Triple-stranded DNA:RNA helices of unknown function in vertebrate mitochondria associate with replication and transcription. Antiparallel Hoogsteen pairings form triplexes at physiological conditions. Intermolecular antiparallel triplexes require inverted 3'-to-5' RNA polymerization, which was never observed. Three rare, long natural 3'-to-5' inverted GenBank RNAs from mice mitochondria suggest occasional inverted transcription, putatively coding for proteins. BLAST aligns 18 GenBank-stored proteins with hypothetical proteins translated from the 3'-to-5' inverted Mus musculus mitochondrial genome. Three are DNA-binding, five are membrane proteins. 25% of main frame codons contribute to their 3'-to-5' overlap coding. Properties of these codons match those of overlap coding protein genes, as compared to codons not expected involved in inverted coding: a) nucleotide contents at synonymous codon positions in mitochondrial genomes fit replicational deamination gradients (A->G and C->T), but digress from gradients when functioning as nonsynonymous positions in putative 3'-to-5' overlapping genes; b) bias against 'circular code' codons (codon groups creating unambiguity between frames), and favouring homogenous codons (AAA, CCC, GGG, TTT) characterize overlapping genes, including putative 3'-to-5' overlapping genes, as compared to nonoverlapping coding sequences from the same main frame gene. This signature correlates with digression from deamination gradients. Deamination and circular code tests confirm independently alignment-based predictions of overlapping 3'-to-5' protein coding genes. Results indicate varying expression for different 3'-to-5' overlapping genes. Inverted 3'-to-5' RNA is produced, perhaps by an unknown RNA polymerase (invertase) putatively coded by 3'-to-5' inverted RNA.

Polymerization of non-complementary RNA: Systematic symmetric nucleotide exchanges mainly involving uracil produce mitochondrial RNA transcripts coding for cryptic overlapping genes

Usual DNA→RNA transcription exchanges T→U. Assuming different systematic symmetric nucleotide exchanges during translation, some GenBank RNAs match exactly human mitochondrial sequences (exchange rules listed in decreasing transcript frequencies): C↔U, A↔U, A↔U+C↔G (two nucleotide pairs exchanged), G↔U, A↔G, C↔G, none for A↔C, A↔G+C↔U, and A↔C+G↔U. Most unusual transcripts involve exchanging uracil. Independent measures of rates of rare replicational enzymatic DNA nucleotide misinsertions predict frequencies of RNA transcripts systematically exchanging the corresponding misinserted nucleotides. Exchange transcripts self-hybridize less than other gene regions, self-hybridization increases with length, suggesting endoribonuclease-limited elongation. Blast detects stop codon depleted putative protein coding overlapping genes within exchange-transcribed mitochondrial genes. These align with existing GenBank proteins (mainly metazoan origins, prokaryotic and viral origins underrepresented). These GenBank proteins frequently interact with RNA/DNA, are membrane transporters, or are typical of mitochondrial metabolism. Nucleotide exchange transcript frequencies increase with overlapping gene densities and stop densities, indicating finely tuned counterbalancing regulation of expression of systematic symmetric nucleotide exchange-encrypted proteins. Such expression necessitates combined activities of suppressor tRNAs matching stops, and nucleotide exchange transcription. Two independent properties confirm predicted exchanged overlap coding genes: discrepancy of third codon nucleotide contents from replicational deamination gradients, and codon usage according to circular code predictions. Predictions from both properties converge, especially for frequent nucleotide exchange types. Nucleotide exchanging transcription apparently increases coding densities of protein coding genes without lengthening genomes, revealing unsuspected functional DNA coding potential.

Systematic asymmetric nucleotide exchanges produce human mitochondrial RNAs cryptically encoding for overlapping protein coding genes

GenBank’s EST database includes RNAs matching exactly human mitochondrial sequences assuming systematic asymmetric nucleotide exchange-transcription along exchange rules: A→G→C→U/T→A (12 ESTs), A→U/T→C→G→A (4 ESTs), C→G→U/T→C (3 ESTs), and A→C→G→U/T→A (1 EST), no RNAs correspond to other potential asymmetric exchange rules. Hypothetical polypeptides translated from nucleotide-exchanged human mitochondrial protein coding genes align with numerous GenBank proteins, predicted secondary structures resemble their putative GenBank homologue’s. Two independent methods designed to detect overlapping genes (one based on nucleotide contents analyses in relation to replicative deamination gradients at third codon positions, and circular code analyses of codon contents based on frame redundancy), confirm nucleotide-exchange-encrypted overlapping genes. Methods converge on which genes are most probably active, and which not, and this for the various exchange rules. Mean EST lengths produced by different nucleotide exchanges are proportional to (a) extents that various bioinformatics analyses confirm the protein coding status of putative overlapping genes; (b) known kinetic chemistry parameters of the corresponding nucleotide substitutions by the human mitochondrial DNA polymerase gamma (nucleotide DNA misinsertion rates); (c) stop codon densities in predicted overlapping genes (stop codon readthrough and exchanging polymerization regulate gene expression by counterbalancing each other). Numerous rarely expressed proteins seem encoded within regular mitochondrial genes through asymmetric nucleotide exchange, avoiding lengthening genomes. Intersecting evidence between several independent approaches confirms the working hypothesis status of gene encryption by systematic nucleotide exchanges.

Overlapping genes coded in the 3′-to-5′-direction in mitochondrial genes and 3′-to-5′ polymerization of non-complementary RNA by an ‘invertase’

Suppressor tRNAs induce expression of additional (off-frame) genes coded by stopless genetic codes without lengthening genomes, decreasing DNA replication costs. RNA 3′-to-5′ polymerization by tRNAHis guanylyltransferase suggests further cryptic code: hypothetical ‘invertases’ polymerizing in the 3′-to-5′ direction, advancing in the 5′-to-3′ direction would produce non-complementary RNA templated by regular genes, with different coding properties. Assuming ‘invertase’ activity, BLAST analyses detect GenBank-stored RNA ESTs and proteins (some potentially coding for the hypothesized invertase) for human mitochondrial genes. These peptides’ predicted secondary structures resemble their GenBank homologues’. 3′-to-5′ EST lengths increase with their self-hybridization potential: Single-stranded RNA degradation perhaps limits 3′-to-5′ elongation. Independent methods confirm predicted 3′-to-5′ overlapping genes: (a) Presumed 3′-to-5′ overlapping genes avoid codons belonging to circular codes; (b) Spontaneous replicational deamination (mutation) gradients occur at 3rd codon positions, unless these are involved in overlap coding, because mutations are counter selected in overlapping genes. Tests a and b converge on predicted 3′-to-5′ gene expression levels. Highly expressed ones include also fewer stops, and mitochondrial genomes (in Primates and Drosophila) adapt to avoid dependence of 3′-to-5′ coding upon antitermination tRNA activity. Secondary structure, circular code, gradient and coevolution analyses yield each clear positive results independently confirming each other. These positive results (including physical evidence for 3′-to-5′ ESTs) indicate that 3′-to-5′ coding and invertase activity is an a priori improbable working hypothesis that cannot be dismissed. Note that RNAs produced by invertases potentially produce triple-stranded DNA:RNA helices by antiparallel Hoogsteen pairings at physiological pH, as previously observed for mitochondrial genomes.

Coding Constraints Modulate Chemically Spontaneous Mutational Replication Gradients in Mitochondrial Genomes

Distances from heavy and light strand replication origins determine duration mitochondrial DNA remains singlestranded during replication. Hydrolytic deaminations from A->G and C->T occur more on single- than doublestranded DNA. Corresponding replicational nucleotide gradients exist across mitochondrial genomes, most at 3rd, least 2nd codon positions. DNA singlestrandedness during RNA transcription causes gradients mainly in long-lived species with relatively slow metabolism (high transcription/replication ratios). Third codon nucleotide contents, evolutionary results of mutation cumulation, follow replicational, not transcriptional gradients in Homo; observed human mutations follow transcriptional gradients. Synonymous third codon position transitions potentially alter adaptive off frame information. No mutational gradients occur at synonymous positions forming off frame stops (these adaptively stop early accidental frameshifted protein synthesis), nor in regions coding for putative overlapping genes according to an overlapping genetic code reassigning stop codons to amino acids. Deviation of 3rd codon nucleotide contents from deamination gradients increases with coding importance of main frame 3rd codon positions in overlapping genes (greatest if these are 2nd position in overlapping genes). Third codon position deamination gradients calculated separately for each codon family are strongest where synonymous transitions are rarely pathogenic; weakest where transitions are frequently pathogenic. Synonymous mutations affect translational accuracy, such as error compensation of misloaded tRNAs by codon-anticodon mismatches (prevents amino acid misinsertion despite tRNA misacylation), a potential cause of pathogenic mutations at synonymous codon positions. Indeed, codon-family-specific gradients are inversely proportional to error compensation associated with gradient-promoted transitions. Deamination gradients reflect spontaneous chemical reactions in singlestranded DNA, but functional coding constraints modulate gradients.

Mitochondrial tRNAs as light strand replication origins: Similarity between anticodon loops and the loop of the light strand replication origin predicts initiation of DNA replication

Stem-loop hairpins formed by mitochondrial light strand replication origins (OL) and by heavy strand DNA coding for tRNAs that form OL-like structures initiate mitochondrial replication. The loops are recognized by one of the two active sites of the vertebrate mitochondrial gamma polymerase, which are homologous to the active sites of class II amino-acyl tRNA synthetases. Therefore, the polymerase site recognizing the OL loop could recognize tRNA anticodon loops and sequence similarity between anticodon and OL loops should predict initiation of DNA replication at tRNAs. Strengths of genome-wide deamination gradients starting at tRNA genes estimate extents by which replication starts at that tRNA. Deaminations (A-->G and C-->T) occur proportionally to time spent single stranded by heavy strand DNA during mitochondrial light strand replication. Results show that deamination gradients starting at tRNAs are proportional to sequence similarity between OL and tRNA loops: most for anticodon-, least D-, intermediate for TpsiC-loops, paralleling tRNA synthetase recognition interactions with these tRNA loops. Structural and sequence similarities with regular OLs predict OL function, loop similarity is dominant in most tRNAs. Analyses of sequence similarity and structure independently substantiate that DNA sequences coding for mitochondrial tRNAs sometimes function as alternative OLs. Pathogenic mutations in anticodon loops increase similarity with the human OL loop, non-pathogenic polymorphisms do not. Similarity/homology alignment hypotheses are experimentally testable in this system.

A comparative approach to elucidate chloroplast genome replication

BackgroundElectron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts.ResultsWe address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the Small Single Copy (SSC) and the Large Single Copy (LSC) regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication.ConclusionSingle-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population

Hybridization between mitochondrial heavy strand tDNA and expressed light strand tRNA modulates the function of heavy strand tDNA as light strand replication origin

Mitochondrial heavy strand (HS) tDNA codes for tRNAs and frequently functions as the light strand (LS) replication origin (OL). During replication, HS sites remain single-stranded until their LS complement is synthesized, a state prone to hydrolytic deaminations of C → T and A → G, causing genome-wide deamination gradients starting at OLs and proportional to time spent single-stranded. Gradient strength is proportional to OL formation by HS tDNAs. Hypothetically, hybridization between HS tDNA and its expressed complement tRNA should decrease OL activity for LS-, but not HS-encoded tRNAs. Comparisons between primate genomes and between pathogenic and non-pathogenic human polymorphisms both confirm corresponding predictions on OL activity. In primates, strengths of deamination gradients starting at tDNAs functioning as OLs and coding for LS tRNAs decrease proportionally to stabilities of HS tDNA-LS tRNA hybridization; not so for HS tRNAs. Similarly, in mutants of human HS tDNAs coding for LS tRNAs, pathogenic mutants of tDNAs usually not forming OLs form weaker HS tDNA-LS tRNA duplexes than non-pathogenic ones; the opposite is true for tDNAs usually forming OLs. No trend was detected for HS tDNA coding for HS tRNA. tDNA-tRNA hybridization of the modal (most frequent) human tDNA sequence is more stable than of other, rarer non-pathogenic polymorphisms, suggesting similar but weaker mutational effects on tDNA/tRNA functions than in pathogenic mutants. HS tDNA-LS tRNA hybridization appears to compete with OL formation by HS tDNA self-hybridization.

Mitochondrial tRNA sequences as unusual replication origins: Pathogenic implications for Homo sapiens

The heavy strand of vertebrate mitochondrial genomes accumulates deaminations proportionally to the time it spends single-stranded during replication. A previous study showed that the strength of genome-wide deamination gradients originating from tRNA gene's locations increases with their capacities to form secondary structures resembling mitochondrial origins of light strand replication (OL), suggesting an alternative function for tRNA sequences. We hypothesize that this function is frequently pathogenic for those tRNA genes that normally do not form OL-like structures, because this could cause excess mutations in genome regions unadapted to tolerate them. In human mitochondrial genomes, pathogenic tRNA variants usually form less OL-like structures than non-pathogenic ones in cases where the normal non-pathogenic tRNA variant can function as OL, as evolutionary analyses reveal. For tRNAs lacking the putative OL-like functioning capacity, pathogenic variants form more OL-like secondary structures, particularly structures that might invoke bi-directional replication (true for 14 among 21 tRNA species, p<0.05, sign test; significantly at p<0.05 (1 tailed test) for 7 tRNA species), but not more unidirectional replication invoking structures. Accounting for the functional cloverleaf-like structure-forming capacities of tRNAs yields similar results. Rare, non-pathogenic tRNA mutants tend to form more OL-like structures than the common, non-pathogenic ones, suggesting weak directional selection also among non-pathogenic variants. The duration spent single stranded by a region of the heavy strand (DssH) during replication, estimated by integrating over all regions that can function as OL in Homo sapiens mitochondrial genomes, increases with distance of that region from the Dloop. This suggests convergence of single-strandedness during replication and transcription, and explains conserved locations of tRNA species in mitochondrial genomes and bacterial operons. These locations minimize deamination costs only in anticodons and not in other tRNA regions, during replication and transcription. Therefore, putative functioning as OLs by tRNA sequences is normal at some locations and pathogenic at others.

Possible multiple origins of replication in primate mitochondria: Alternative role of tRNA sequences

DNA replication in vertebrate mitochondria is usually directional, leaving different portions of the genome single-stranded for different periods of time. During this time, mutations resulting from deaminations of cytosines to thymines and adenines to guanines accumulate on the heavy strand. Therefore, T/C and G/A ratios increase along mitochondrial genomes, proportionally to the time spent single-stranded during replication. Such trends exist at third codon positions for base ratios averaged across genes in individual genomes as well as for gene-specific and site-specific substitution frequencies estimated using phylogenetic methods. We use multiple regressions to test for the potential functioning of all 12 tRNA clusters in 19 primate mitochondrial genomes as alternative origins of light strand replication (OL). We provide a general algorithm for calculating time spent single stranded by a given site for any possible locations of the site and OL. For codon positions 1, 2, and 3, respectively, 23%, 9% and 35% of tRNA gene clusters have significant ( p < 0.0 5 ) deamination gradients originating from them. The strength of the deamination gradient originating from tRNA gene clusters varies among species, and for five clusters, correlates with the tendency of tRNA genes in each of these clusters to form secondary structures that resemble the OL's structure. This is notably true for all codon positions for tRNA-Lys, which in absence of nuclear regulation, forms secondary structures resembling the hairpin structure of OL. For two tRNA gene clusters, correlations were statistically significant, but opposite to the direction expected by the known unidirectional replication, putatively compatible with bi-directional replication. Few substitutions in tRNA sequences can be neutral at the level of cloverleaf structure and function, yet significantly alter capacities to form OL-like structures, causing sudden evolution of genome-wide nucleotide contents.