Publications by authors named "Howard Gamper"

41 Publications

Twice exploration of tRNA +1 frameshifting in an elongation cycle of protein synthesis.

Nucleic Acids Res 2021 Aug 20. Epub 2021 Aug 20.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with -1 frameshifting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab734DOI Listing
August 2021

Loss of -methylation of G37 in tRNA induces ribosome stalling and reprograms gene expression.

Elife 2021 08 12;10. Epub 2021 Aug 12.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, United States.

-methylation of G37 is required for a subset of tRNAs to maintain the translational reading-frame. While loss of mG37 increases ribosomal +1 frameshifting, whether it incurs additional translational defects is unknown. Here, we address this question by applying ribosome profiling to gain a genome-wide view of the effects of mG37 deficiency on protein synthesis. Using as a model, we show that mG37 deficiency induces ribosome stalling at codons that are normally translated by mG37-containing tRNAs. Stalling occurs during decoding of affected codons at the ribosomal A site, indicating a distinct mechanism than that of +1 frameshifting, which occurs after the affected codons leave the A site. Enzyme- and cell-based assays show that mG37 deficiency reduces tRNA aminoacylation and in some cases peptide-bond formation. We observe changes of gene expression in mG37 deficiency similar to those in the stringent response that is typically induced by deficiency of amino acids. This work demonstrates a previously unrecognized function of mG37 that emphasizes its role throughout the entire elongation cycle of protein synthesis, providing new insight into its essentiality for bacterial growth and survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.70619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384417PMC
August 2021

Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation.

Nat Commun 2021 07 30;12(1):4644. Epub 2021 Jul 30.

RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, UMass Medical School, Worcester, MA, USA.

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-24911-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324841PMC
July 2021

Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA.

Nat Commun 2021 01 12;12(1):328. Epub 2021 Jan 12.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA.

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-20373-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803779PMC
January 2021

Purification and Use of tRNA for Enzymatic Post-translational Addition of Amino Acids to Proteins.

STAR Protoc 2020 Dec 9;1(3):100207. Epub 2020 Dec 9.

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Post-translational addition of amino acids to proteins by enzymes using aminoacyl-tRNA is an emerging regulatory mechanism. Examples include Arg transfer in eukaryotes, Leu/Phe transfer in bacteria, and tRNA-synthetase-mediated addition of amino acids to Lys side chains. Here, we present a method of purification and use of tRNA for such reactions, focusing on tRNA and its use for arginylation. This method can also be used for other tRNA-mediated reactions. For complete details on the use and execution of this protocol, please refer to Avcilar-Kucukgoze et al. (2020).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xpro.2020.100207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757669PMC
December 2020

A Label-Free Assay for Aminoacylation of tRNA.

Genes (Basel) 2020 10 7;11(10). Epub 2020 Oct 7.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Aminoacylation of tRNA generates an aminoacyl-tRNA (aa-tRNA) that is active for protein synthesis on the ribosome. Quantification of aminoacylation of tRNA is critical to understand the mechanism of specificity and the flux of the aa-tRNA into the protein synthesis machinery, which determines the rate of cell growth. Traditional assays for the quantification of tRNA aminoacylation involve radioactivity, either with a radioactive amino acid or with a [3'-P]-labeled tRNA. We describe here a label-free assay that monitors aminoacylation by biotinylation-streptavidin (SA) conjugation to the α-amine or the α-imine of the aminoacyl group on the aa-tRNA. The conjugated aa-tRNA product is readily separated from the unreacted tRNA by a denaturing polyacrylamide gel, allowing for quantitative measurement of aminoacylation. This label-free assay is applicable to a wide range of amino acids and tRNA sequences and to both classes of aminoacylation. It is more sensitive and robust than the assay with a radioactive amino acid and has the potential to explore a wider range of tRNA than the assay with a [3'-P]-labeled tRNA. This label-free assay reports kinetic parameters of aminoacylation quantitatively similar to those reported by using a radioactive amino acid, suggesting its broad applicability to research relevant to human health and disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes11101173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601589PMC
October 2020

tRNA-Derived Fragments Can Serve as Arginine Donors for Protein Arginylation.

Cell Chem Biol 2020 07 16;27(7):839-849.e4. Epub 2020 Jun 16.

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Arginyltransferase ATE1 mediates posttranslational arginylation and plays key roles in multiple physiological processes. ATE1 utilizes arginyl (Arg)-tRNA as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we address the question of ATE1- Arg-tRNA specificity as a potential mechanism enabling this competition in vivo. Using in vitro arginylation assays and Ate1 knockout models, we find that, in addition to full-length tRNA, ATE1 is also able to utilize short tRNA fragments that bear structural resemblance to tRNA-derived fragments (tRF), a recently discovered class of small regulatory non-coding RNAs with global emerging biological role. Ate1 knockout cells show a decrease in tRF generation and a significant increase in the ratio of tRNA:tRF compared with wild type, suggesting a functional link between tRF and arginylation. We propose that generation of physiologically important tRFs can serve as a switch between translation and protein arginylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2020.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409373PMC
July 2020

Codon-Specific Translation by mG37 Methylation of tRNA.

Front Genet 2018 10;9:713. Epub 2019 Jan 10.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, United States.

Although the genetic code is degenerate, synonymous codons for the same amino acid are not translated equally. Codon-specific translation is important for controlling gene expression and determining the proteome of a cell. At the molecular level, codon-specific translation is regulated by post-transcriptional epigenetic modifications of tRNA primarily at the wobble position 34 and at position 37 on the 3'-side of the anticodon. Modifications at these positions determine the quality of codon-anticodon pairing and the speed of translation on the ribosome. Different modifications operate in distinct mechanisms of codon-specific translation, generating a diversity of regulation that is previously unanticipated. Here we summarize recent work that demonstrates codon-specific translation mediated by the mG37 methylation of tRNA at CCC and CCU codons for proline, an amino acid that has unique features in translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fgene.2018.00713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335274PMC
January 2019

Author Correction: The selective tRNA aminoacylation mechanism based on a single G•U pair.

Nature 2018 12;564(7736):E37

RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.

In Fig. 1b of this Article, a U was inadvertently inserted after G15 in the D loop. The original Article has not been corrected.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0760-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550308PMC
December 2018

tRNA 3'-amino-tailing for stable amino acid attachment.

RNA 2018 12 14;24(12):1878-1885. Epub 2018 Sep 14.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

Amino acids are attached to the tRNA 3'-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3'-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3'-amino-3'-deoxy A76 in a 3'-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown. Here we compare five different tailing protocols in parallel, all dependent on the CCA-adding enzyme [CTP(ATP): tRNA nucleotidyl transferase; abbreviated as the CCA enzyme] to exchange the natural ribose with the modified one. We show that the most efficient protocol is achieved by the CCA-catalyzed pyrophosphorolysis removal of the natural A76 in equilibrium with the addition of the appropriate ATP analog to synthesize the modified 3'-end. This protocol for 3'-amino-tailing affords quantitative and stable attachment of a broad range of amino acids to tRNA, indicating its general utility for studies of aminoacyl-tRNAs in both canonical and noncanonical activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.068015.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239183PMC
December 2018

Selective terminal methylation of a tRNA wobble base.

Nucleic Acids Res 2018 04;46(7):e37

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3'-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo5U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo5U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo5U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNAPro and tRNAVal for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNAPro inactivates the terminal methylation, whereas introduction of G35 to tRNAVal confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNAPro, the terminal methylation at U34 is dependent on the primary m1G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909439PMC
April 2018

Transcription-translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits.

Nucleic Acids Res 2017 Nov;45(19):11043-11055

Department of Biochemistry, University of California, Riverside, CA 92521, USA.

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkx719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737488PMC
November 2017

Effect of Nascent Peptide Steric Bulk on Elongation Kinetics in the Ribosome Exit Tunnel.

J Mol Biol 2017 06 5;429(12):1873-1888. Epub 2017 May 5.

Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

All proteins are synthesized by the ribosome, a macromolecular complex that accomplishes the life-sustaining tasks of faithfully decoding mRNA and catalyzing peptide bond formation at the peptidyl transferase center (PTC). The ribosome has evolved an exit tunnel to host the elongating new peptide, protect it from proteolytic digestion, and guide its emergence. It is here that the nascent chain begins to fold. This folding process depends on the rate of translation at the PTC. We report here that besides PTC events, translation kinetics depend on steric constraints on nascent peptide side chains and that confined movements of cramped side chains within and through the tunnel fine-tune elongation rates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2017.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511029PMC
June 2017

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Nucleic Acids Res 2017 04;45(7):4081-4093

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkw1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397188PMC
April 2017

A novel HSD17B10 mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression.

RNA Biol 2016 05 7;13(5):477-85. Epub 2016 Mar 7.

d Department of Biochemistry and Molecular Biology , Thomas Jefferson University , Philadelphia , PA , USA.

We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5'-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15476286.2016.1159381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962811PMC
May 2016

The UGG Isoacceptor of tRNAPro Is Naturally Prone to Frameshifts.

Int J Mol Sci 2015 Jul 1;16(7):14866-83. Epub 2015 Jul 1.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Native tRNAs often contain post-transcriptional modifications to the wobble position to expand the capacity of reading the genetic code. Some of these modifications, due to the ability to confer imperfect codon-anticodon pairing at the wobble position, can induce a high propensity for tRNA to shift into alternative reading frames. An example is the native UGG isoacceptor of E. coli tRNAPro whose wobble nucleotide U34 is post-transcriptionally modified to cmo5U34 to read all four proline codons (5'-CCA, 5'-CCC, 5'-CCG, and 5'-CCU). Because the pairing of the modified anticodon to CCC codon is particularly weak relative to CCA and CCG codons, this tRNA can readily shift into both the +1 and +2-frame on the slippery mRNA sequence CCC-CG. We show that the shift to the +2-frame is more dominant, driven by the higher stability of the codon-anticodon pairing at the wobble position. Kinetic analysis suggests that both types of shifts can occur during stalling of the tRNA in a post-translocation complex or during translocation from the A to the P-site. Importantly, while the +1-frame post complex is active for peptidyl transfer, the +2-frame complex is a poor peptidyl donor. Together with our recent work, we draw a mechanistic distinction between +1 and +2-frameshifts, showing that while the +1-shifts are suppressed by the additional post-transcriptionally modified m1G37 nucleotide in the anticodon loop, the +2-shifts are suppressed by the ribosome, supporting a role of the ribosome in the overall quality control of reading-frame maintenance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms160714866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519876PMC
July 2015

Maintenance of protein synthesis reading frame by EF-P and m(1)G37-tRNA.

Nat Commun 2015 May 26;6:7226. Epub 2015 May 26.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA.

Maintaining the translational reading frame poses difficulty for the ribosome. Slippery mRNA sequences such as CC[C/U]-[C/U], read by isoacceptors of tRNA(Pro), are highly prone to +1 frameshift (+1FS) errors. Here we show that +1FS errors occur by two mechanisms, a slow mechanism when tRNA(Pro) is stalled in the P-site next to an empty A-site and a fast mechanism during translocation of tRNA(Pro) into the P-site. Suppression of +1FS errors requires the m(1)G37 methylation of tRNA(Pro) on the 3' side of the anticodon and the translation factor EF-P. Importantly, both m(1)G37 and EF-P show the strongest suppression effect when CC[C/U]-[C/U] are placed at the second codon of a reading frame. This work demonstrates that maintaining the reading frame immediately after the initiation of translation by the ribosome is an essential aspect of protein synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms8226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445466PMC
May 2015

Loss-of-function alanyl-tRNA synthetase mutations cause an autosomal-recessive early-onset epileptic encephalopathy with persistent myelination defect.

Am J Hum Genet 2015 Apr 26;96(4):675-81. Epub 2015 Mar 26.

Medical Scientist Training Program, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Center for Genetic Medicine Research, Children's National Health System, Washington, DC 20010, USA; Departments of Integrated Systems Biology and of Pediatrics, George Washington University, Washington, DC 20052, USA. Electronic address:

Mutations in genes encoding aminoacyl-tRNA synthetases are known to cause leukodystrophies and genetic leukoencephalopathies-heritable disorders that result in white matter abnormalities in the central nervous system. Here we report three individuals (two siblings and an unrelated individual) with severe infantile epileptic encephalopathy, clubfoot, absent deep tendon reflexes, extrapyramidal symptoms, and persistently deficient myelination on MRI. Analysis by whole exome sequencing identified mutations in the nuclear-encoded alanyl-tRNA synthetase (AARS) in these two unrelated families: the two affected siblings are compound heterozygous for p.Lys81Thr and p.Arg751Gly AARS, and the single affected child is homozygous for p.Arg751Gly AARS. The two identified mutations were found to result in a significant reduction in function. Mutations in AARS were previously associated with an autosomal-dominant inherited form of axonal neuropathy, Charcot-Marie-Tooth disease type 2N (CMT2N). The autosomal-recessive AARS mutations identified in the individuals described here, however, cause a severe infantile epileptic encephalopathy with a central myelin defect and peripheral neuropathy, demonstrating that defects of alanyl-tRNA charging can result in a wide spectrum of disease manifestations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2015.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385183PMC
April 2015

Post-transcriptional modifications to tRNA--a response to the genetic code degeneracy.

RNA 2015 Apr;21(4):642-4

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.049825.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371315PMC
April 2015

A divalent metal ion-dependent N(1)-methyl transfer to G37-tRNA.

Chem Biol 2014 Oct 11;21(10):1351-1360. Epub 2014 Sep 11.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10(th) Street, BLSB 220, Philadelphia, PA 19107, USA. Electronic address:

The catalytic mechanism of the majority of S-adenosyl methionine (AdoMet)-dependent methyl transferases requires no divalent metal ions. Here we report that methyl transfer from AdoMet to N(1) of G37-tRNA, catalyzed by the bacterial TrmD enzyme, is strongly dependent on divalent metal ions and that Mg(2+) is the most physiologically relevant. Kinetic isotope analysis, metal rescue, and spectroscopic measurements indicate that Mg(2+) is not involved in substrate binding, but in promoting methyl transfer. On the basis of the pH-activity profile indicating one proton transfer during the TrmD reaction, we propose a catalytic mechanism in which the role of Mg(2+) is to help to increase the nucleophilicity of N(1) of G37 and stabilize the negative developing charge on O(6) during attack on the methyl sulfonium of AdoMet. This work demonstrates how Mg(2+) contributes to the catalysis of AdoMet-dependent methyl transfer in one of the most crucial posttranscriptional modifications to tRNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2014.07.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224600PMC
October 2014

The selective tRNA aminoacylation mechanism based on a single G•U pair.

Nature 2014 Jun 11;510(7506):507-11. Epub 2014 Jun 11.

1] RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan [2] Department of Biophysics and Biochemistry and Laboratory of Structural Biology, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan [3] RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.

Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNA(Ala) selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNA(Ala) with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3'-CCA region. Thus, the 3'-CCA region of tRNA(Ala) with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature13440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323281PMC
June 2014

Amino acid-dependent stability of the acyl linkage in aminoacyl-tRNA.

RNA 2014 Jun 21;20(6):758-64. Epub 2014 Apr 21.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

Aminoacyl-tRNAs are the biologically active substrates for peptide bond formation in protein synthesis. The stability of the acyl linkage in each aminoacyl-tRNA, formed through an ester bond that connects the amino acid carboxyl group with the tRNA terminal 3'-OH group, is thus important. While the ester linkage is the same for all aminoacyl-tRNAs, the stability of each is not well characterized, thus limiting insight into the fundamental process of peptide bond formation. Here, we show, by analysis of the half-lives of 12 of the 22 natural aminoacyl-tRNAs used in peptide bond formation, that the stability of the acyl linkage is effectively determined only by the chemical nature of the amino acid side chain. Even the chirality of the side chain exhibits little influence. Proline confers the lowest stability to the linkage, while isoleucine and valine confer the highest, whereas the nucleotide sequence in the tRNA provides negligible contribution to the stability. We find that, among the variables tested, the protein translation factor EF-Tu is the only one that can protect a weak acyl linkage from hydrolysis. These results suggest that each amino acid plays an active role in determining its own stability in the acyl linkage to tRNA, but that EF-Tu overrides this individuality and protects the acyl linkage stability for protein synthesis on the ribosome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.044123.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024630PMC
June 2014

The temperature sensitivity of a mutation in the essential tRNA modification enzyme tRNA methyltransferase D (TrmD).

J Biol Chem 2013 Oct 28;288(40):28987-96. Epub 2013 Aug 28.

From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m(1)G37 on the 3' side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.485797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789996PMC
October 2013

Conservation of structure and mechanism by Trm5 enzymes.

RNA 2013 Sep 25;19(9):1192-9. Epub 2013 Jul 25.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

Enzymes of the Trm5 family catalyze methyl transfer from S-adenosyl methionine (AdoMet) to the N¹ of G37 to synthesize m¹ G37-tRNA as a critical determinant to prevent ribosome frameshift errors. Trm5 is specific to eukaryotes and archaea, and it is unrelated in evolution from the bacterial counterpart TrmD, which is a leading anti-bacterial target. The successful targeting of TrmD requires detailed information on Trm5 to avoid cross-species inhibition. However, most information on Trm5 is derived from studies of the archaeal enzyme Methanococcus jannaschii (MjTrm5), whereas little information is available for eukaryotic enzymes. Here we use human Trm5 (Homo sapiens; HsTrm5) as an example of eukaryotic enzymes and demonstrate that it has retained key features of catalytic properties of the archaeal MjTrm5, including the involvement of a general base to mediate one proton transfer. We also address the protease sensitivity of the human enzyme upon expression in bacteria. Using the tRNA-bound crystal structure of the archaeal enzyme as a model, we have identified a single substitution in the human enzyme that improves resistance to proteolysis. These results establish conservation in both the catalytic mechanism and overall structure of Trm5 between evolutionarily distant eukaryotic and archaeal species and validate the crystal structure of the archaeal enzyme as a useful model for studies of the human enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.039503.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753926PMC
September 2013

Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position.

J Mol Biol 2013 Oct 28;425(20):3888-906. Epub 2013 May 28.

Department of Chemistry, Portland State University, PO Box 751, Portland, OR 97207, USA; Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97239, USA.

The 2-thiouridine (s(2)U) at the wobble position of certain bacterial and eukaryotic tRNAs enhances aminoacylation kinetics, assists proper codon-anticodon base pairing at the ribosome A-site, and prevents frameshifting during translation. By mass spectrometry of affinity-purified native Escherichia coli tRNA1(Gln)UUG, we show that the complete modification at the wobble position 34 is 5-carboxyaminomethyl-2-thiouridine (cmnm(5)s(2)U). The crystal structure of E. coli glutaminyl-tRNA synthetase (GlnRS) bound to native tRNA1(Gln) and ATP demonstrates that cmnm(5)s(2)U34 improves the order of a previously unobserved 11-amino-acid surface loop in the distal β-barrel domain of the enzyme and imparts other local rearrangements of nearby amino acids that create a binding pocket for the 2-thio moiety. Together with previously solved structures, these observations explain the degenerate recognition of C34 and modified U34 by GlnRS. Comparative pre-steady-state aminoacylation kinetics of native tRNA1(Gln), synthetic tRNA1(Gln) containing s(2)U34 as sole modification, and unmodified wild-type and mutant tRNA1(Gln) and tRNA2(Gln) transcripts demonstrates that the exocyclic sulfur moiety improves tRNA binding affinity to GlnRS 10-fold compared with the unmodified transcript and that an additional fourfold improvement arises from the presence of the cmnm(5) moiety. Measurements of Gln-tRNA(Gln) interactions at the ribosome A-site show that the s(2)U modification enhances binding affinity to the glutamine codons CAA and CAG and increases the rate of GTP hydrolysis by E. coli EF-Tu by fivefold.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2013.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521407PMC
October 2013

Potential for interdependent development of tRNA determinants for aminoacylation and ribosome decoding.

Nat Commun 2011 ;2:329

Thomas Jefferson University, Department of Biochemistry and Molecular Biology, Philadelphia, Pennsylvania 19107, USA.

Although the nucleotides in tRNA required for aminoacylation are conserved in evolution, bacterial aminoacyl-transfer RNA synthetases are unable to acylate eukaryotic tRNA. The cross-species barrier may be due to the absence of eukaryote-specific domains from bacterial aminoacyl-transfer RNA synthetases. Here we show that whereas Escherichia coli CysRS cannot acylate human tRNA(Cys), the fusion of a eukaryote-specific domain of human CysRS overcomes the cross-species barrier in human tRNA(Cys). In addition to enabling recognition of the sequence differences in the tertiary core of tRNA(Cys), the fused eukaryotic domain redirects the specificity of E. coli CysRS from the A37 present in bacterial tRNA(Cys) to the G37 in mammals. Further experiments show that the accuracy of codon recognition on the ribosome was also highly sensitive to the A37G transition in tRNA(Cys). These results raise the possibility of the development of tRNA nucleotide determinants for aminoacylation being interdependent with those for ribosome decoding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms1331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799875PMC
September 2011

Distinct kinetic determinants for the stepwise CCA addition to tRNA.

RNA 2009 Oct 20;15(10):1827-36. Epub 2009 Aug 20.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

The universally conserved CCA sequence is present at the 3' terminal 74-76 positions of all active tRNA molecules as a functional tag to participate in ribosome protein synthesis. The CCA enzyme catalyzes CCA synthesis in three sequential steps of nucleotide addition at rapid and identical rates. However, the kinetic determinant of each addition is unknown, thus limiting the insights into the kinetic basis of CCA addition. Using our recently developed single turnover kinetics of Escherichia coli CCA enzyme as a model, we show here that the identical rate of the stepwise CCA addition is determined by distinct kinetic parameters. Specifically, the kinetics of C74 and C75 addition is controlled by the chemistry of nucleotidyl transfer, whereas the kinetics of A76 addition is controlled by a prechemistry conformational transition of the active site. In multiple turnover condition, all three steps are controlled by slow product release, indicating enzyme processivity from one addition to the next. However, the processivity decreases as the enzyme progresses to complete the CCA synthesis. Together, these results suggest the existence of a network of diverse kinetic parameters that determines the overall rate of CCA addition for tRNA maturation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.1669109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743048PMC
October 2009

Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine.

Nucleic Acids Res 2008 Dec 5;36(22):6999-7008. Epub 2008 Nov 5.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkn797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602760PMC
December 2008

RecA-mediated strand invasion of DNA by oligonucleotides substituted with 2-aminoadenine and 2-thiothymine.

Nucleic Acids Res 2008 Dec 25;36(21):6806-15. Epub 2008 Oct 25.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Sequence-specific recognition of DNA is a critical step in gene targeting. Here we describe unique oligonucleotide (ON) hybrids that can stably pair to both strands of a linear DNA target in a RecA-dependent reaction with ATP or ATPgammaS. One strand of the hybrids is a 30-mer DNA ON that contains a 15-nt-long A/T-rich central core. The core sequence, which is substituted with 2-aminoadenine and 2-thiothymine, is weakly hybridized to complementary locked nucleic acid or 2'-OMe RNA ONs that are also substituted with the same base analogs. Robust targeting reactions took place in the presence of ATPgammaS and generated metastable double D-loop joints. Since the hybrids had pseudocomplementary character, the component ONs hybridized less strongly to each other than to complementary target DNA sequences composed of regular bases. This difference in pairing strength promoted the formation of joints capable of accommodating a single mismatch. If similar joints can form in vivo, virtually any A/T-rich site in genomic DNA could be selectively targeted. By designing the constructs so that the DNA ON is mismatched to its complementary sequence in DNA, joint formation might allow the ON to function as a template for targeted point mutation and gene correction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkn755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2588519PMC
December 2008

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3' end.

RNA 2008 Oct 28;14(10):2245-53. Epub 2008 Aug 28.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

All mature tRNA molecules have the conserved CCA sequence at the 3' end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 3' end. Using Escherichia coli tRNA(Cys) as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNA(Cys), separated from the unlabeled precursor tRNA by reverse phase high-pressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNA(Cys) with E. coli CysRS reveals an equilibrium K(d) closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyC-labeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.1158508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553749PMC
October 2008
-->