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PubMed1997

The path of mRNA through the bacterial ribosome: a site-directed crosslinking study using new photoreactive derivatives of guanosine and uridine.

Two new photoreactive nucleotide derivatives have been applied in site-directed crosslinking studies with mRNA analogues. 6-Thioguanosine triphosphate or 5-methyleneaminouridine triphosphate was incorporated into mRNA analogues by T7 transcription; after transcription, the 5-methyleneaminouridine residues were converted to a diazirine derivative. mRNA analogues carrying either 6-thioguanosine or the diazirine derivative were bound to Escherichia coli ribosomes in the presence of tRNA(f)(Met), and photo-crosslinking was induced by irradiation at 350 nm. With 6-thioguanosine, specific crosslinks were observed from downstream positions +8 or +9 of the mRNA to nt 1196 in helix 34 of the 16S rRNA, and from position +12 to nt 530 in helix 18. With the diazirine derivative, a crosslink from position +2 (within the AUG codon) to nt 926 in helix 28 was found. Taken together with previous data obtained from downstream sites in mRNA analogues carrying 4-thiouridine residues, specific crosslinks have now been identified from downstream mRNA positions +2, +4, +6, +7, +8, +9, +11, and +12. The data confirm that the three 16S rRNA regions involved-helices 18, 28, and 34-are in the direct neighborhood of the decoding area of the 30S subunit.

Bacteriophage T7↗
PubMed2006

Proteomic approaches to dissect platelet function: Half the story.

Platelets play critical roles in diverse hemostatic and pathologic disorders and are broadly implicated in various biological processes that include inflammation, wound healing, and thrombosis. Recent progress in high-throughput mRNA and protein profiling techniques has advanced our understanding of the biological functions of platelets. Platelet proteomics has been adopted to decode the complex processes that underlie platelet function by identifying novel platelet-expressed proteins, dissecting mechanisms of signal or metabolic pathways, and analyzing functional changes of the platelet proteome in normal and pathologic states. The integration of transcriptomics and proteomics, coupled with progress in bioinformatics, provides novel tools for dissecting platelet biology. In this review, we focus on current advances in platelet proteomic studies, with emphasis on the importance of parallel transcriptomic studies to optimally dissect platelet function. Applications of these global profiling approaches to investigate platelet genetic diseases and platelet-related disorders are also addressed.

Animals↗
PubMed1992

Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function.

Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637]. Here we show that mutation of C1501 to G was equally inactivating but that the double mutant C1401:G1501 with the base pair reversed had virtually full activity for tRNA binding to the P, A, and I sites and for peptide bond formation. Initiation-dependent formation of the first peptide bond remained 70-85% inhibited, despite full 70S initiation complex formation ability as evidenced by the ability to form fMET-puromycin. These results suggest that the defect in formation of the first peptide bond lies in filling the initial A site, Ai, rather than the subsequent elongation A sites, Ae. An increased mobility around the anticodon was detected by UV cross-linking of the anticodon of P-site-bound tRNA to C1399 as well as to the expected C1400. These findings provide the first experimental evidence for the existence of the G1401:C1501 base pair and show that this base pair, located at the decoding site, is essential for function. The structural implications of tertiary base pair formation are discussed.

Base Sequence↗
PubMed2004

Plant genomics: the third wave.

Completing the primary genomic sequence of Arabidopsis thaliana was a major milestone, being the first plant genome and only the third high-quality finished eukaryotic genome sequence. Understanding how the genome sequence comprehensively encodes developmental programs and environmental responses is the next major challenge for all plant genome projects. This requires fully characterizing the genes, the regulatory sequences, and their functions. We discuss several functional genomics approaches to decode the linear sequence of the reference plant Arabidopsis thaliana, including full-length cDNA collections, microarrays, natural variation, knockout collections, and comparative sequence analysis. Genomics provides the essential tools to speed the work of the traditional molecular geneticist and is now a scientific discipline in its own right.

Arabidopsis↗
PubMed1996

On the mechanism of leftward frameshifting at several hungry codons.

We have used lacZ reporter genes to assess leftward ribosome frameshifting on sequences containing the quadruplet U UUC followed by several different triplets coding for lysine, isoleucine, or leucine. Limitation for lysine-tRNA provokes leftward frameshifting when the slippery quadruplet is followed by either lysine codon aag or aaa, but not when followed by an isoleucine or leucine codon. Limitation for isoleucine provokes frameshifting when the quadruplet is followed by either isoleucine codon aua or auc, but not when it is followed by a lysine codon. We conclude that the quadruplet promotes shifting when the ribosome is stalled at any "hungry" codon immediately after it. Changing the quadruplet to U AGC, at which peptidyl-tRNA cognate to the AGC triplet will be mismatched at all three anticodon positions if it slips left, abolishes frameshifting when the ribosome is stalled at the next position. We conclude that the U UUC quadruplet promotes frameshifting by virtue of its ability to pair with a left-slipped peptidyl-tRNA. The frameshift promoted by isoleucine-tRNA limitation of the U UUC aua sequence was analyzed by amino acid sequencing of the protein product. It occurs through reading of the Cau histidine codon overlapping the hungry codon from the left. This result rules out a "simultaneous slippage" type of mechanism. It strongly suggests instead that starvation-promoted frameshifting occurs primarily by slippage of peptidyl-tRNA just upstream of the stall site, followed by decoding of the triplet overlapping the stall site from the left or 5' side. A secondary finding is that the last base of the "hungry" codon has a moderate effect on its shiftiness, aag being shiftier than aaa, and aua being shiftier than auc.

Amino Acid Sequence↗
PubMed1998

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

Aldehydes↗
PubMed2006

Local decoding of sequences and alignment-free comparison.

Subword composition plays an important role in a lot of analyses of sequences. Here we define and study the "local decoding of order N of sequences," an alternative that avoids some drawbacks of "subwords of length N" approaches while keeping informations about environments of length N in the sequences ("decoding" is taken here in the sense of hidden Markov modeling, i.e., associating some state to all positions of the sequence). We present an algorithm for computing the local decoding of order N of a given set of sequences. Its complexity is linear in the total length of the set (whatever the order N) both in time and memory space. In order to show a use of local decoding, we propose a very basic dissimilarity measure between sequences which can be computed both from local decoding of order N and composition in subwords of length N. The accuracies of these two dissimilarities are evaluated, over several datasets, by computing their linear correlations with a reference alignment-based distance. These accuracies are also compared to the one obtained from another recent alignment-free comparison.

Computational Biology↗
PubMed2026

Accelerating inference in genomic and proteomic foundation models via speculative decoding.

MOTIVATION: Genomic and protein foundation models (GFMs and PFMs) have demonstrated strong performance in learning the language of DNA and proteins, but their use in large-scale sequence generation is limited by the latency of autoregressive decoding. Because every token triggers a forward pass of a large Transformer, whose inference is relatively slow, long-sequence generation quickly becomes costly. RESULTS: In this work we adapt speculative decoding to a representative GFM: the DNA model DNAGPT and two representative PFMs: ProGen2 and ProtGPT2. We implement a probabilistic variant of speculative decoding, in which a lightweight draft model proposes short token spans and a larger target model verifies or corrects them in parallel, while preserving the target model's sampling distribution. Across all three models we systematically study the effect of speculation window length, temperature, draft architecture and prompt length, and we benchmark tokens per second over multiple runs per configuration. Speculative decoding yields consistent speedups over standard key-value cached decoding, with maximum observed speedup reaching 100% increase, while average gains across models ranging between 20% and 40% (e.g. 1.2×-1.4×), without changing the underlying target model predictions. Our results show that speculative decoding is a practical and model-agnostic strategy for accelerating genomic and proteomic sequence generation without sacrificing prediction quality. AVAILABILITY AND IMPLEMENTATION: All code and results are freely available at https://github.com/Georgakopoulos-Soares-lab/BioSpecDec.

Genomics↗
PubMed1995

Comparisons of ribosomal RNA sequences from amitochondrial protozoa: implications for processing, mRNA binding and paromomycin susceptibility.

The amitochondrial (a-mt) protozoa include four groups of organisms that are of interest as important human parasites and as probable descendents of the earliest branches of eukaryotic evolution. These organisms have not been directly compared in terms of structure and function of a specific molecule. We sequenced portions of their rRNA-encoding genes coding for the internal transcribed spacers (ITS1 and 2) and adjoining small subunit (SS), 5.8S and large subunit (LS) rRNAs. Included are sites for RNA processing, mRNA interaction and aminoglycoside binding, as well as potential protein-encoding genes. The ITS of all a-mt protozoa examined are relatively short, but otherwise diverse. They include one or two predominant nucleotides (A in Entamoeba and Trichomonas, T in Encephalitozoon and C in Giardia) and have minimal potential secondary structure, which may form the basis for the preferential processing of ITS sequences. The mechanism employed by a-mt protozoa to bind mRNA may be unique, since Giardia, Trichomonas and Entamoeba mRNAs have usually short 5' non-coding regions. In bacteria, the 3' terminus of the SS rRNA is involved in mRNA binding; analysis of Entamoeba and Trichomonas mRNA 5' non-coding sequences suggests an analogous mechanism involving potential base pairing to the loop of the terminal SS rRNA hairpin. Giardia sensitivity to paromomycin was previously correlated with the presence of a C:G bp near the decoding region of SS rRNA. This bp is also present in Entamoeba and Trichomonas, consistent with their susceptibility. Its absence in Encephalitozoon and other microsporidia predicts paromomycin resistance, and suggests a distinct evolutionary origin for this group.

Amino Acid Sequence↗
PubMed2005

The first position of a codon placed in the A site of the human 80S ribosome contacts nucleotide C1696 of the 18S rRNA as well as proteins S2, S3, S3a, S30, and S15.

Messenger RNA analogues (42-mers) containing a GAC codon (aspartic acid) in the middle of their sequence followed by a s(4)UGA stop codon were used to identify the components of the human ribosomal A site in direct contact with the photoactivatable 4-thiouridine (s(4)U) residue. We compared the behavior of the nonphased ribosome-mRNA complex, (-)tRNA(Asp), to the one of the phased complex, (+)tRNA(Asp), in the absence and in the presence of eRF1, the eukaryotic class 1 translation termination factor of human origin. The patterns of cross-links obtained for the three complexes were similar to those previously reported for rabbit ribosomes [Chavatte, L., et al. (2001) Eur. J. Biochem. 268, 2896-2904]. Cross-links involving proteins S2, S3, S3a, and S30 were poorly dependent on the presence of tRNA(Asp) and eRF1. Cross-linking to nucleotide C1696 of 18S rRNA occurred in all complexes, but its yield was at least two times higher in the phased complex with an empty A site than in the nonphased complex or when the A site was occupied by eRF1. In contrast, protein S15 cross-linked only in the phased complex in the absence of eRF1. The data obtained point to notable differences in organization of the decoding site between mammalian and prokaryotic ribosomes and to large internal mobility of the components of the tRNA (eRF1)-free A site.

Base Sequence↗
PubMed2000

Structure of the 30S ribosomal subunit.

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

Bacterial Proteins↗
PubMed2025

Integrated genomics and morphological approach reveals interspecific gene flow cases and decodes the origin of selected feathergrasses (Poaceae, Stipa).

Central Asia is a diversity hotspot of arid-adapted grasses from the genus Stipa, with approximately 100 taxa found in the region. Recent studies in the steppe areas of Kazakhstan revealed specimens displaying intermediate morphology, distinguishing them from other taxa that grow sympatrically. Using integrative taxonomy, we investigated whether these individuals resulted from natural speciation or hybridisation, and if so, we would like to know which species were involved in this process feathergrasses. Research conducted in steppes of central Kazakhstan (Kyzylorda region), revealed the existence of individuals morphologically intermediate between S. arabica and S. richteriana, suggesting that these are probably of hybrid origin. Morphology and SNP markers validated the specimens as F1 hybrid between the aforementioned species by cladding separately based on neighbor-joining phylogenetic tree. Moreover, genetic structure displayed a separate cluster and showed almost equal genetic admixture between S. arabica and S. richteriana. Additionally, fastStructure analysis detected two geographically separated cryptic genotypes within S. richteriana population and their involvement in the hybridisation resulted in occurrence of S. × heptapotamica, S. × czerepanovii and S. × korshinskyi which recently were suggested as hybrids. Based on these evidences, we described a new nothospecies S. × kyzylordensis, as F1 hybrid. Furthermore, morphologically, the nothospecies delimited with other hybrids in Kazakh steppe area, marking the first report of hybridisation between S. arabica and S. richteriana, along with molecular evidence for the origin of further species supposed to be hybrids. This finding is crucial to understanding species diversity and hybridisation process in morphologically and genetically distant Stipa species.

Poaceae↗
PubMed2005

Torsional restraint: a new twist on frameshifting pseudoknots.

mRNA pseudoknots have a stimulatory function in programmed -1 ribosomal frameshifting (-1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for -1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168-174]. A separate 'torsional restraint' model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115-1127]. Here, experiments using a series of 'pseudo-pseudoknots' having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that -1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot.

Base Sequence↗
PubMed1999

An extra tRNAGly(U*CU) found in ascidian mitochondria responsible for decoding non-universal codons AGA/AGG as glycine.

Amino acid assignments of metazoan mitochondrial codons AGA/AGG are known to vary among animal species; arginine in Cnidaria, serine in invertebrates and stop in vertebrates. We recently found that in the mitochondria of the ascidian Halocynthia roretzi these codons are exceptionally used for glycine, and postulated that they are probably decoded by a tRNA(UCU). In order to verify this notion unambig-uously, we determined the complete RNA sequence of the mitochondrial tRNA(UCU) presumed to decode codons AGA/AGG in the ascidian mitochondria, and found it to have an unidentified U derivative at the anticodon first position. We then identified the amino acids attached to the tRNA(U*CU), as well as to the conventional tRNAGly(UCC) with an unmodified U34, in vivo. The results clearly demonstrated that glycine was attached to both tRNAs. Since no other tRNA capable of decoding codons AGA/AGG has been found in the mitochondrial genome, it is most probable that this tRNA(U*CU) does actually translate codons AGA/AGG as glycine in vivo. Sequencing of tRNASer(GCU), which is thought to recognize only codons AGU/AGC, revealed that it has an unmodified guanosine at position 34, as is the case with vertebrate mitochondrial tRNASer(GCU) for codons AGA/AGG. It was thus concluded that in the ascidian, codons AGU/AGC are read as serine by tRNASer(GCU), whereas AGA/AGG are read as glycine by an extra tRNAGly(U*CU). The possible origin of this unorthodox genetic code is discussed.

Animals↗
PubMed2026

Genome-scale perturb-seq in primary human CD4+ T cells maps context-specific regulators of T cell programs and human immune traits.

Gene regulatory networks encode the fundamental logic of cellular functions, but systematic network mapping remains challenging, especially in cell states relevant to human biology and disease. Here, we perturbed all expressed genes across 22 million primary human CD4+ T cells from four donors and developed a probe-based perturb-seq platform to measure the transcriptome effects in cells at rest and after stimulation. These data allowed us to map genes regulating immune pathways, including previously uncharacterized regulators of cytokine production. Importantly, active regulators and the gene programs they control changed dramatically across stimulation conditions. Perturbation signatures enabled us to model T cell states observed in population-scale transcriptomic atlases, nominating regulators of T cell polarization and of age-related phenotypes. Finally, we leveraged perturb-seq to implicate context-specific gene regulatory pathways in autoimmune disease risk. Our study provides a foundational resource and new approaches to decode T cell function and human immune traits.

CD4(+) T cell polarization↗
PubMed1992

Selenocysteyl-tRNAs recognize UGA in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus.

Selenocysteyl-tRNAs that decode UGA were previously identified in representatives of three of the five life kingdoms which were the monera, animal and protist kingdoms. In the present study, we show that these tRNAs also occur in representatives of the two remaining kingdoms, plants and fungi; i.e., selenocysteyl-tRNAs which code for UGA occur in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus. The fact that selenocysteyl-tRNAs are present in all five life kingdoms strongly suggests that UGA, in addition to dictating the cessation of protein synthesis, also codes for selenocysteine in the universal genetic code.

Base Sequence↗
PubMed1971

Alteration of a 30S ribosomal protein accompanying the ram mutation in Escherichia coli.

The functional peculiarities of ram mutants correlate with an observed alteration in chromatographic mobility of P4(a), a specific protein of the 30S ribosomal subunit. This finding is supported by ribosomal reconstitution experiments. These facts, together with the known location of the ram mutational site in the vicinity of other 30S genetic determinants, suggest that ram is the structural gene for P4(a). The known contrasting roles of ram and strA in determining translational efficiency require that the function of P4(a) should be explained in relation to P10 (the 30S-subunit protein defined by strA). One consequence of altering P4(a), a key protein in ribosome assembly, might be to change the interaction of P10 with the 30S subunit. The functional interrelationship of P4(a) and P10 is discussed in terms of the possible roles of these two proteins in regulating access of tRNA molecules to the decoding site.

Autoradiography↗
PubMed1990

Decoding positional information: regulation of the pair-rule gene hairy.

In the series of local gene activations that occur during early Drosophila development, the striped expression patterns of the pair-rule genes provide the first indication of segmental periodicity. The experiments that we report here address the question of how these patterns arise, by studying the regulation of one of these genes, hairy. We show that each of the seven stripes of hairy expression is controlled by a distinct subset of cis-acting regulatory elements, some mediating transcriptional activation and others transcriptional repression. In general, elements necessary and sufficient for triggering a particular stripe response are clustered on the DNA and appear to overlap or be interspersed with elements involved in at least one other stripe response. Our results extend previous findings suggesting that periodic hairy expression arises by a decoding process in which each stripe is triggered by particular combinations or concentrations of regulatory factors. These regulatory factors are likely to include the products of the gap class of segmentation genes that are required for activating or positioning particular subsets of hairy stripes and are expressed with overlapping distributions during early embryogenesis.

Animals↗