About

Uttam RajBhandary is the Lester Wolfe Professor of Molecular Biology Emeritus at MIT. Before closing his lab, he studied interactions between RNAs and proteins, focusing on gene expression and gene regulation. His research examined tRNA structure, function, and biosynthesis using biochemistry, genetics, and in vivo functional analyses. His work contributed to understanding RNA-protein interactions, which play a crucial role in gene expression and development.

Research topics

• Biology
• Biochemistry
• Genetics
• Molecular biology
• Chemistry

Selected publications

• Khorana, Har Gobind

  Elsevier eBooks · 2022-01-01

  book-chapterSenior author
  PublisherDOI

• Mitochondrial methionyl N-formylation affects steady-state levels of oxidative phosphorylation complexes and their organization into supercomplexes

  Journal of Biological Chemistry · 2018-08-07 · 24 citations

  articleOpen access

  N-Formylation of the Met-tRNAMet by the nuclearly encoded mitochondrial methionyl-tRNA formyltransferase (MTFMT) has been found to be a key determinant of protein synthesis initiation in mitochondria. In humans, mutations in the MTFMT gene result in Leigh syndrome, a progressive and severe neurometabolic disorder. However, the absolute requirement of formylation of Met-tRNAMet for protein synthesis in mammalian mitochondria is still debated. Here, we generated a Mtfmt-KO mouse fibroblast cell line and demonstrated that N-formylation of the first methionine via fMet-tRNAMet by MTFMT is not an absolute requirement for initiation of protein synthesis. However, it differentially affected the efficiency of synthesis of mtDNA-coded polypeptides. Lack of methionine N-formylation did not compromise the stability of these individual subunits but had a marked effect on the assembly and stability of the OXPHOS complexes I and IV and on their supercomplexes. In summary, N-formylation is not essential for mitochondrial protein synthesis but is critical for efficient synthesis of several mitochondrially encoded peptides and for OXPHOS complex stability and assembly into supercomplexes.

  PublisherDOI

• Initiation of protein synthesis from a termination codon (gene regulation/initiation with glutamine/initiation with AUC/chloramphenicol acetyltransferase gene/amber mutants)

  2016-01-01

  articleSenior author

  We show that the amber termination codon UAG can initiate protein synthesis in Escherichia coli. We mutated the initiation codon AUG of the chloramphenicol acetyltransferase (CAT) gene to UAG (CATaml) and translated mRNA derived from the mutant CAT gene in E. coli S-30 extracts. A full-length CAT polypeptide was synthesized in the presence of tRNAfmJt, a mutant E. coli initiator tRNA which has a change in the anticodon sequence from CAU to CUA. Addition of purified E. coli glutaminyl-tRNA synthetase sub- stantially stimulated synthesis of the CAT polypeptide. Thus, initiation of protein synthesis with UAG and tRNAfm9t most likely occurs with glutamine and not methionine. The UAG codon also initiates protein synthesis in vivo. To eliminate a weak secondary site of initiation from AUC, the fifth codon, we further mutagenized the CATaml gene at codons 2 (GAG -- GAC) and 5 (AUC -- ACC). Transformation of E. coli with the resultant CATaml.2.5 gene yielded transformants that syn- thesized CAT polypeptide and were resistant to chloramphen- icol only when they were also transformed with the mutant tRNAfmet gene. Immunoblot analyses and assays for CAT enzyme activity in extracts from transformed cells indicate that initiation from UAG is efficient, 60-70% of that obtained from AUG. Initiation of protein synthesis from UAG using a mutant initiator tRNA allows tightly regulated expression of specific genes. This may be generally useful for overproduction in E. coli and other eubacteria of proteins which are toxic to these

  Publisher

• Correction: Corrigendum: Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

  Nature Communications · 2016-01-21 · 146 citations

  erratumOpen access

  Nature Communications 6: Article number: 7520 (2015); Published: 3 July 2015; Updated: 21 January 2016. The authors inadvertently omitted Sumit Jaiswal, who was originally included in the Acknowledgements section of this Article and contributed to the cloning of transgenic constructs and the balancing of transgenic flies, from the author list.

  PublisherOA PDFDOI

• (tRNA identity switch/methionine tRNA/glutamine tRNA/formylation/ribosomal P site)

  2016-01-01

  articleSenior author

  We show that the two most important prop- erties needed for a tRNA to function in initiation in Escherichia coli are its ability to be formylated and its ability to bind to the ribosomal P site. This conclusion is based on conversion of two different elongator tRNAs to ones that can act as initiators in E. coli. We transplanted the features unique to E. coli and eubacterial initiator tRNAs to E. coli elongator methionine tRNA (tRNAMet) along with an anticodon sequence change and analyzed their activities in initiation in E. coli. Introduction of a CloA72 mismatch at the end of the acceptor stem of tRNAMet, which generates the minimal features necessary for formyla- tion, produces a tRNA with very low activity in initiation. Subsequent introduction of three consecutive G-C base pairs at the bottom of the anticodon stem, which is necessary for ribosomal P site binding, produces a tRNA with significant activity in initiation. Furthermore, introduction of the features necessary for formylation and for ribosomal P site binding into E. coli elongator glutamine tRNA produces a tRNA that initiates protein synthesis in E. coli.

  Publisher

• Interesting and unusual features in crassa mitochondrial tyrosine tranm (tRNA tertiary structure/mitochondrial evolution/in vitro labelin

  2016-01-01

  articleSenior author

  The mitochondrial tyrosine tRNA from Neu- rospora crassa has been sequenced and found to have several interesting features: (i) It resembles prokaryotic rather than eukaryotic tyrosine tRNAs in that it possesses a large variable loop (loop III); moreover, it can be quantitatively aminoacylated by Escherichia coli tyrosyl-tRNA synthetase but not by yeast tyrosyl-tRNA synthetase. (ii) This tRNA differs from all tRNAs sequenced to date in lacking the A residue at position 14 and the constant purine residue at position 15, two nucleosides that have been found so far in loop I of all tRNAs and that have been implicated in base-base tertiary interactions, respectively, with the universal U residue at position 8 and the constant pyrimidine residue at the end of loop III. (iii) Unlike the N. crassa mito- chondrial initiator tRNA, this tRNA contains the usual TIC sequence in loop IV and the highly conserved GG sequence in loop I common to other tRNAs. Many features of tRNA structure have been strongly conserved through evolution. Initiator tRNAs in particular demonstrate this conservation. Although initiator tRNAs from both pro- karyotes and eukaryotic cytoplasm possess specific sequence features that distinguish each class from the other and from most noninitiator tRNAs, in terms of overall nucleotide se- quence the seventeen or so initiator tRNAs sequenced display a high degree of sequence homology (65-100%). The recent structural analysis of an initiator tRNA from the mitochondria of Neurospora crassa (1) presented an interesting exception to this pattern. Not only did this methionine tRNA differ greatly in its sequence from other initiators, both eukaryotic and prokaryotic, but it also lacked some structural features here- tofore found in all tRNAs that are active in protein synthesis (2). This unexpected finding generated several interesting questions. Are the structural features noted in the mitochondrial initiator tRNA unique to the initiator species? Will noninitiator tRNAs in the mitochondrion also show less stringent adherence to known patterns of tRNA structure and evolution compared to the known prokaryotic and eukaryotic tRNAs? If such dif- ferences do exist, do they reflect some variation in the functional demands placed upon mitochondrial tRNAs as compared to

  Publisher

• Identification of YbeY-Protein Interactions Involved in 16S rRNA Maturation and Stress Regulation in Escherichia coli

  mBio · 2016-11-09 · 62 citations

  articleOpen access

  YbeY is part of a core set of RNases in Escherichia coli and other bacteria. This highly conserved endoribonuclease has been implicated in several important processes such as 16S rRNA 3' end maturation, 70S ribosome quality control, and regulation of mRNAs and small noncoding RNAs, thereby affecting cellular viability, stress tolerance, and pathogenic and symbiotic behavior of bacteria. Thus, YbeY likely interacts with numerous protein or RNA partners that are involved in various aspects of cellular physiology. Using a bacterial two-hybrid system, we identified several proteins that interact with YbeY, including ribosomal protein S11, the ribosome-associated GTPases Era and Der, YbeZ, and SpoT. In particular, the interaction of YbeY with S11 and Era provides insight into YbeY's involvement in the 16S rRNA maturation process. The three-way association between YbeY, S11, and Era suggests that YbeY is recruited to the ribosome where it could cleave the 17S rRNA precursor endonucleolytically at or near the 3' end maturation site. Analysis of YbeY missense mutants shows that a highly conserved beta-sheet in YbeY-and not amino acids known to be important for YbeY's RNase activity-functions as the interface between YbeY and S11. This protein-interacting interface of YbeY is needed for correct rRNA maturation and stress regulation, as missense mutants show significant phenotypic defects. Additionally, structure-based in silico prediction of putative interactions between YbeY and the Era-30S complex through protein docking agrees well with the in vivo results. IMPORTANCE: Ribosomes are ribonucleoprotein complexes responsible for a key cellular function, protein synthesis. Their assembly is a highly coordinated process of RNA cleavage, RNA posttranscriptional modification, RNA conformational changes, and protein-binding events. Many open questions remain after almost 5 decades of study, including which RNase is responsible for final processing of the 16S rRNA 3' end. The highly conserved RNase YbeY, belonging to a core set of RNases essential in many bacteria, was previously shown to participate in 16S rRNA processing and ribosome quality control. However, detailed mechanistic insight into YbeY's ribosome-associated function has remained elusive. This work provides the first evidence that YbeY is recruited to the ribosome through interaction with proteins involved in ribosome biogenesis (i.e., ribosomal protein S11, Era). In addition, we identified key residues of YbeY involved in the interaction with S11 and propose a possible binding mode of YbeY to the ribosome using in silico docking.

  PublisherOA PDFDOI

• Nonsense suppression in archaea

  Proceedings of the National Academy of Sciences · 2015-04-27 · 7 citations

  articleOpen accessSenior author

  Bacterial strains carrying nonsense suppressor tRNA genes played a crucial role in early work on bacterial and bacterial viral genetics. In eukaryotes as well, suppressor tRNAs have played important roles in the genetic analysis of yeast and worms. Surprisingly, little is known about genetic suppression in archaea, and there has been no characterization of suppressor tRNAs or identification of nonsense mutations in any of the archaeal genes. Here, we show, using the β-gal gene as a reporter, that amber, ochre, and opal suppressors derived from the serine and tyrosine tRNAs of the archaeon Haloferax volcanii are active in suppression of their corresponding stop codons. Using a promoter for tRNA expression regulated by tryptophan, we also show inducible and regulatable suppression of all three stop codons in H. volcanii. Additionally, transformation of a ΔpyrE2 H. volcanii strain with plasmids carrying the genes for a pyrE2 amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in the pyrE2 gene can be complemented by expression of the amber suppressor tRNA. These results pave the way for generating archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in archaeal and archaeviral genetics. We also provide possible explanations for why suppressor tRNAs have not been identified in archaea.

  PublisherOA PDFDOI

• Essentiality of threonylcarbamoyladenosine (t⁶<scp>A</scp>), a universal t<scp>RNA</scp> modification, in bacteria

  Molecular Microbiology · 2015-09-04 · 96 citations

  articleOpen access

  Threonylcarbamoyladenosine (t(6)A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t(6)A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t(6)A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNA(Ile)-lysidine synthetase. We confirm that t(6)A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t(6)A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t(6)A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t(6)A(-) D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t(6)A in tRNAs. Thus, although t(6)A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.

  PublisherOA PDFDOI

• Essentiality of threonylcarbamoyladenosine (t[superscript 6]A), a universal tRNA modification, in bacteria

  DSpace@MIT (Massachusetts Institute of Technology) · 2015-09-01

  articleOpen accessSenior author

  Threonylcarbamoyladenosine (t[superscript 6]A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t6A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t[superscript 6]A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNA[superscript Ile]-lysidine synthetase. We confirm that t6A is essential in Escherichia coli and a survey of genomewide essentiality studies shows that genes for t[superscrip 6]A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t[superscript 6]A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t6A- D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t[superscript 6]A in tRNAs. Thus, although t[superscript 6]A is universally conserved in tRNAs, its role in translation might vary greatly between organisms.

  Publisher

Recent grants

• NIH Grant R01GM046942

  NIH · $670k · 1998

• Structure and Function of Transfer Ribonucleic Acids

  NIH · $7.0M · 1978–2016

• NIH Grant R56GM017151

  NIH · $559k · 2010

• NIH Grant R01GM067741

  NIH · $1.1M · 2010

• NIH Grant R37GM017151

  NIH · $5.0M · 2009

Frequent coauthors

• Phillip A. Sharp

  Massachusetts Institute of Technology

  45 shared

• Frank A. Laski

  41 shared

• Caroline Köhrer

  Moderna Therapeutics (United States)

  32 shared

• Peter Palese

  29 shared

• Mario R. Capecchi

  University of Utah

  28 shared

• R Belagaje

  28 shared

• Robert M. Hudziak

  Sarepta Therapeutics (United States)

  26 shared

• Grenville Norton

  University of Nottingham

  25 shared

Awards & honors

• Fellow, American Academy of Arts and Sciences (1991)