Frameshifting as a tool in analysis of transfer RNA modification and translation Ramunė Leipuvienė Department of Molecular Biology Umeå University Umeå 2004 Doctoral Dissertation 2004 Department of Molecular Biology Umeå University SE-901 87 Umeå, Sweden Copyright © 2004 by Ramunė Leipuvienė ISBN 91-7305-709-6 Printed by Solfjädern Offset AB Umeå, Sweden 2 CONTENTS ABSTRACT ..........................................................................................................................5 MAIN PAPERS ....................................................................................................................7 INTRODUCTION ................................................................................................................8 TRANSLATION AND READING FRAME MAINTENANCE ..................................................... 8 Ribosomes ......................................................................................................................8 Types of translational errors .........................................................................................9 Reading frame maintenance errors ............................................................................10 Spontaneous frameshifting and cell physiology.........................................................12 Genetics of translational frame maintenance ............................................................12 Programmed frameshifting .........................................................................................13 Ribosomal hopping ......................................................................................................15 TRNA PROPERTIES ..........................................................................................................15 Structure of tRNAs ......................................................................................................15 tRNA abundance and codon frequency......................................................................17 tRNA aminoacylation specificity.................................................................................17 Function of modified nucleosides...............................................................................18 IRON-SULFUR CLUSTERS AND TRNA THIOLATION .........................................................19 Iron-sulfur cluster proteins .........................................................................................19 Synthesis of iron-sulfur clusters .................................................................................21 Other proteins involved in iron-sulfur cluster metabolism........................................23 ISC machinery and tRNA thiolation...........................................................................24 AIMS OF THIS WORK ....................................................................................................28 RESULTS AND DISCUSSION.........................................................................................29 FRAMESHIFTING INDUCED BY DECREASED STABILITY AND REDUCED AMINOACYLATION OF A MINOR ARGININE TRNA (PAPER I)...........................................30 Arg tRNA 5 The sufF mutant has an altered mnm UCU ..........................................................30 Arg Arg tRNA 5 tRNA 5 mnm UCU and/or arginyl- mnm UCU levels are decreased in the mutants.....31 Glu tRNA 5 2 Peptidyl- mnm s UUC frameshifts when the ribosome is pausing..........................32 3 RIBOSOME SLIPPAGE INDUCED BY DEFECTS IN THE RIBOSOMAL PROTEIN L9 (PAPER II)..........................................................................................................................33 Frameshift mutation is suppressed by mutations in ribosomal protein L9...............33 The mechanism of ribosomal slippage induced by L9 mutants is unknown.............34 ENZYMES REQUIRED FOR TRNA THIOLATION (PAPERS III AND IV)................................35 Formation of s2C and ms2io6A in tRNA requires functional iron-sulfur proteins ..35 TtcA is involved in thiolation of cytidine at position 32 and may hold an iron- sulfur cluster ...............................................................................................................37 Lack of IscA specifically decreases the levels of ms2io6A but not that of s2C ..........37 Two pathways for sulfur in tRNA thiolation .............................................................38 tRNA thiolations and frameshifting............................................................................41 CONCLUSIONS.................................................................................................................43 ACKNOWLEDGEMENTS ...............................................................................................44 REFERENCES ...................................................................................................................45 PAPERS I-IV 4 ABSTRACT Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene Arg Arg tRNA 5 tRNA 5 coding for the minor mnm UCU . The altered mnm UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA- Arg tRNA 5 AGA frameshifting site the altered mnm UCU reads the AGA codon Glu tRNA 5 2 inefficiently. This induces a ribosomal pause, allowing the mnm s UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated. During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated 5 nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site. 6 MAIN PAPERS This thesis contains the following publications referred to by their Roman numerals: I. Decreased stability and reduced aminoacylation of a minor arginine tRNA mediates ribosomal frameshifting. Ramune Leipuviene and Glenn R. Björk. (2004), Manuscript. II. Ribosome slippage induced by mutations in the ribosomal protein L9. Ramune Leipuviene and Glenn R. Björk. (2004), Manuscript. III. Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways. Ramune Leipuviene, Qiang Qian, and Glenn R. Björk. (2004), Journal of Bacteriology, 186:758-766. IV. The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium. Gunilla Jäger, Ramune Leipuviene, Michael G. Pollard, Qiang Qian, and Glenn R. Björk. (2004), Journal of Bacteriology, 186:750-757. 7 INTRODUCTION Translation and reading frame maintenance Ribosomes One of the most important processes in the cell is the conversion of genetic information into proteins. This process is initiated by transcription of information contained in the DNA to messenger RNA (mRNA), which later is translated into proteins. Translation occurs via the synchronized action of ribosomes, mRNA, aminoacylated transfer RNAs, and a huge number of different protein factors. Large ribonucleoprotein particles termed ribosomes are the main players in translation. The bacterial 70S ribosome consists of small (30S) and large (50S) subunits. The 30S subunit comprises 16S rRNA (1542 nucleotides) and 21 proteins, and the 50S subunit comprises 23S rRNA (2904 nucleotides), 5S rRNA (120 nucleotides), and 36 proteins (Gao et al., 2003). The interface between the subunits is occupied by tRNAs that bind to the A (aminacyl), P (peptidyl), and E (exit) ribosomal binding sites. The A site accepts the incoming aminoacylated tRNA, while the P site holds the tRNA with the nascent peptide chain, and the E site possibly holds the deacylated tRNA before