Directed Mutations Recode Mitochondrial Genes: From Regular to Stopless Genetic Codes

Mitochondrial genetic codes evolve as side effects of stop codon ambiguity: suppressor tRNAs with anticodons translating stops transform genetic codes to stopless genetic codes. This produces peptides from frames other than regular ORFs, potentially increasing protein numbers coded by single sequences. Previous descriptions of marine turtle Olive Ridley mitogenomes imply directed stop-depletion of noncoding +1 gene frames, stop-creation recodes regular ORFs to stopless genetic codes. In this analysis, directed stop codon depletion in usually noncoding gene frames of the spiraling whitefly Aleurodicus dispersusʼ mitogenome produces new ORFs, introduces stops in regular ORFs, and apparently increases coding redundancy between different gene frames. Directed stop codon mutations switch between peptides coded by regular and stopless genetic codes. This process seems opposite to directed stop creation in HIV ORFs within genomes of immunized elite HIV controllers. Unknown DNA replication/edition mechanisms probably direct stop creation/depletion beyond natural selection on stops. Switches between genetic codes regulate translation of different gene frames

Structural and biochemical characterization of ribosome small subunit-dependent GTPase A (RsgA) from Pseudomonas aeruginosa

The increase in antibiotic resistance among pathogenic bacterial strains presents a significant health threat. The main efforts to combat antibiotic resistance are focused on the development of new antibiotics targeting protein biosynthesis. Ribosome, the large molecular machine responsible for this process, and proteins involved in the translational process represent ideal targets of molecules with antibacterial activity. The ribosome assembly in vivo is an intricate and finely tuned process promoted by the action of several proteins acting as assembly factors, whose precise role is still largely unknown. Small GTPases represent the largest class of ribosome assembly factors in bacteria and are emerging as possible targets to be explored for the development of novel antibacterial strategies. Among them, of particular interest is the Ribosome small subunit-dependent GTPase A (RsgA). RsgA is a late-stage ribosome biogenesis factor involved in the 30S subunit maturation, broadly conserved among bacteria but absent in eukaryotes. RsgA is a circulary permutated GTPase that belongs to an interesting class of GTPases, termed HAS-GTPase, that lack the conserved catalytic glutamine. The circularly permutated GTP binding site is flanked by an OB-fold domain at the N-terminus and by a zinc binding domain at the C-terminus. Despite the large amount of biochemical, structural and genetic data on RsgA achieved in the last decade, its mechanism of action is still not completely understood. Here we focus on the structural and functional characterisation of RsgA from the human pathogenic bacterium Pseudomonas aeruginosa (PaRsgA). The main goal of this work is the determination of the PaRsgA structure by X-ray crystallography. To date, no structure is available for RsgA from this opportunistic pathogen. This knowledge will allow investigate the molecular features for the recognition of GDP and GTP as well as the key determinants for the mechanism of GTP hydrolysis. Moreover, an accurate kinetic analysis of PaRsgA interaction with GDP and GTP, together with a detailed functional characterization of PaRsgA, provided the determination of substrates affinity and biochemical parameters of GTP hydrolysis. The results obtained will pave the way for future experiments aimed at the characterization of the binding mechanism underlying ribosome recognition and to get key insight the GTPase activity of PaRsgA in the presence of other assembly factors and/or the ribosomal particle

Extensive frameshift at all AGG and CCC codons in the mitochondrial cytochrome c oxidase subunit 1 gene of Perkinsus marinus (Alveolata; Dinoflagellata)

Diverse mitochondrial (mt) genetic systems have evolved independently of the more uniform nuclear system and often employ modified genetic codes. The organization and genetic system of dinoflagellate mt genomes are particularly unusual and remain an evolutionary enigma. We determined the sequence of full-length cytochrome c oxidase subunit 1 (cox1) mRNA of the earliest diverging dinoflagellate Perkinsus and show that this gene resides in the mt genome. Apparently, this mRNA is not translated in a single reading frame with standard codon usage. Our examination of the nucleotide sequence and three-frame translation of the mRNA suggest that the reading frame must be shifted 10 times, at every AGG and CCC codon, to yield a consensus COX1 protein. We suggest two possible mechanisms for these translational frameshifts: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons or specialized tRNAs recognizing non-triplet codons, AGGY and CCCCU. Regardless of the mechanism, active and efficient machinery would be required to tolerate the frameshifts predicted in Perkinsus mitochondria. To our knowledge, this is the first evidence of translational frameshifts in protist mitochondria and, by far, is the most extensive case in mitochondria

Structural analysis of stalled ribosomal complexes and their respective rescue mechanisms by Cryo-Electron Microscopy

The ribosome is a multifunctional ribonucleoprotein complex responsible for the translation of the genetic code into proteins. It consists of two subunits, the small ribosomal subunit and the large ribosomal subunit. During initiation of translation, both subunits join and form a functional 70S ribosome that is capable of protein synthesis. In the course of elongation, the ribosome synthesizes proteins according to the codons on the mRNA until it encounters a stop codon leading to the recruitment of release factors 1 or 2 followed by release of the nascent chain. Upon release of the polypeptide chain the subunits dissociate from each other and can be recruited for another round of translation. There are two scenarios that interfere with active translation, namely the formation of so called ‘non-stop’ or ‘no-go’ complexes. In both cases, ribosomes pause translation and without interference of additional factors, they would become stalled. Accumulation of such events leads to a decrease of ribosomal subunits that can be recruited for translation, ultimately resulting in the death of the cell. Using cryo-electron microscopy (cryo-EM), we obtained the structure of alternative rescue factor A (ArfA) together with release factor 2 bound to a ‘non-stop’ complex. Our reconstructions showed that the C-terminal domain of ArfA occupies the empty mRNA channel on the SSU, whereas the N-terminal domain provides a platform for recruiting RF2 in a stop codon-independent way. Thereby, ArfA stabilizes a unique conformation of the switch loop of RF2, responsible for directing the catalytically important GGQ motif towards the PTC. The high-resolution structure of ArfA allowed us to compare its mode of action with trans-translation and alternative rescue factor B, two other factors operating on ‘non-stop’ complexes. A second project focused on elongation factor P (EF-P), a factor that alleviates stalling on polyproline stalled ribosomes. Applying cryo-EM, we were able to show that in the absence of EF-P, the nascent chain is destabilized as the polyproline moiety attached to the P-tRNA is not able to accommodate within the ribosomal tunnel. Binding of modified EF-P to the polyproline stalled complex stabilizes the P-site tRNA and especially the CCA, thereby forcing the nascent chain to adopt an alternative conformation that is favorable for translation to proceed

Detection of frameshifts and improving genome annotation

We developed a new program called GeneTack for ab initio frameshift detection in intronless protein-coding nucleotide sequences. The GeneTack program uses a hidden Markov model (HMM) of a genomic sequence with possibly frameshifted protein-coding regions. The Viterbi algorithm nds the maximum likelihood path that discriminates between true adjacent genes and a single gene with a frameshift. We tested GeneTack as well as two other earlier developed programs FrameD and FSFind on 17 prokaryotic genomes with frameshifts introduced randomly into known genes. We observed that the average frameshift prediction accuracy of GeneTack, in terms of (Sn+Sp)/2 values, was higher by a signicant margin than the accuracy of the other two programs. GeneTack was used to screen 1,106 complete prokaryotic genomes and 206,991 genes with frameshifts (fs-genes) were identifed. Our goal was to determine if a frameshift transition was due to (i) a sequencing error, (ii) an indel mutation or (iii) a recoding event. We grouped 102,731 genes with frameshifts (fs-genes) into 19,430 clusters based on sequence similarity between their protein products (fs-proteins), conservation of predicted frameshift position, and its direction. While fs-genes in 2,810 clusters were classied as conserved pseudogenes and fs-genes in 1,200 clusters were classied as hypothetical pseudogenes, 5,632 fs-genes from 239 clusters pos- sessing conserved motifs near frameshifts were predicted to be recoding candidates. Experiments were performed for sequences derived from 20 out of the 239 clusters; programmed ribosomal frameshifting with eciency higher than 10% was observed for four clusters. GeneTack was also applied to 1,165,799 mRNAs from 100 eukaryotic species and 45,295 frameshifts were identied. A clustering approach similar to the one used for prokaryotic fs-genes allowed us to group 12,103 fs-genes into 4,087 clusters. Known programmed frameshift genes were among the obtained clusters. Several clusters may correspond to new examples of dual coding genes. We developed a web interface to browse a database containing all the fs-genes predicted by GeneTack in prokaryotic genomes and eukaryotic mRNA sequences. The fs-genes can be retrieved by similarity search to a given query sequence, by fs- gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, programmed frameshifts etc. All the tools and the database of fs-genes are available at the GeneTack web site http://topaz.gatech.edu/GeneTack/PhDCommittee Chair: Borodovsky, Mark; Committee Member: Baranov, Pavel; Committee Member: Hammer, Brian; Committee Member: Jordan, King; Committee Member: Konstantinidis, Kostas; Committee Member: Song, L

Platination Kinetics: Insight Into Rna-Cisplatin Interactions As A Probe For Rna Microenvironments

RNAs are crucial for many cellular functions. Thus, studying ligand-RNA interactions and their dynamics in response to changes in the surrounding environment is important. In spite of the well-known DNA coordination, current research also indicates cisplatin binding to RNA. Kinetic studies of rRNA platination reactions are largely unexplored. This research was conducted to achieve two objectives. First, a broad kinetic study was carried out to investigate the cisplatin-rRNA interactions. The structure, function, and ligand interactions depend on RNA microenvironments. Second, the application of platination kinetics as a tool to interrogate RNA electrostatic environments was explored. Three model rRNA hairpins from E. coli ribosome were selected. Two helix 69 (H69) constructs, modified H69 (with pseudouridine) and unmodified H69 (without pseudouridine), and the 790 loop, which has an identical size and nucleotide composition to unmodified H69, were used. Prior to kinetic studies, cisplatin targets on each RNA were determined using RNase T1 mapping combined with MALDI MS, and dimethyl sulfate (DMS) probing. The kinetic studies were carried out under pseudo-first-order conditions and electrostatic properties were evaluated using Brønsted-Debye-Hückel and polyelectrolyte theories. RNase T1 mapping with MALDI MS and dimethyl sulfate (DMS) probing revealed GpG sites as cisplatin targets on RNA. The DMS probing further revealed platination-induced structural changes in RNA. Both the RNA sequence and modified nucleotides showed an impact on platination rates. Kinetic data showed that the platination rate is dependent on cations and the abundance of active cisplatin complexes. Structure, pseudouridylation, availability of active cisplatin species, and cation/Pt+ electrostatic competitions all impact platination of the two H69 RNAs. Probing neomycin-H69 interactions by platination kinetics indicated that structural changes in modified H69 upon aminoglycoside binding could also impact the platination kinetics. Electrostatic models revealed that nucleotide sequence, cations, and H+ ions impact the global RNA electrostatics. The similar global electrostatic properties between the two H69 RNAs indicated that structure-dependent electrostatic changes in modified H69 could be limited to the loop region. In conclusion, this thesis work showed that both intrinsic RNA characteristics such as structure, sequence, and dynamics, as well as bulk solution conditions (e.g. cations and pH), impact cisplatin-RNA interactions. The RNA electrostatic parameters determined in this thesis work illustrated platination kinetics can be used as an informatory tool for probing dynamic RNA microenvironments

Isolation And Analysis Of Peptides Binding To Helix 69 Of E. Coli 23 S Rrna From M13 Phage Display

Peptides binding to helix 69 of domain IV or residues 1906-1924, of 23 S rRNA of E.coli were being selected from a heptapeptide phage library. An experimental system including biotin labeling of RNA and then affinity selection through four rounds was being followed. After sequencing phage clones of the fourth round, two peptide sequences dominated the phage pool, STYTSVS and NQVANHQ. The later sequence was a unique sequence since this sequence contained an abundance of amino acid residues that are also present in the ribosome recycling factor, RRF, and known to make contacts with H69. Phage-display methodology demonstrated the rapid feasibility of identification, and isolation of small peptides that bind to 23 S rRNA in an effort to discover new RNA-binding motifs that have potential therapeutic applications. For evaluating the preliminary binding affinity of these peptides with H69, fluorescence assays were applied. For this assay, the fluorescence intensity of the NQVANHQ Tentagel beads was observed to be higher than STYTSVS Tentagel beads indicating that peptide NQVANHQ is having higher affinity for H69 as compared to STYTSVS peptide. But the higher binding affinity of the NQVANHQ peptide was further validated with more sensitive method of electrospray ionization (ESI) mass spectroscopy. The apparent dissociation constant (Kd) obtained for H69 and NQVANHQ-NH2 peptide was in the low micromolar range (11 µM). This value is comparable to that of aminoglycoside antibiotics binding to the A-site RNA (1 to 10 µM). The ESI-MS experiments with H69 variant UUU RNA and peptide NQVANHQ-NH2 gave the relative dissociation constant (Kd) at 1:1 stoichiometry as 19 µM. The higher value of Kd for this complex revealed that the presence of all three pseudouridine residues positively contributed towards binding of this peptide to H69. Consecutively, to learn about the role of individual pseudouridines at position 1911 and 1915 towards binding of the peptide, the ESI-MS experiments were performed with two H69 variants,UYY and YUY. The apparent dissociation constants (Kd) for the 1:1 complex for these two RNAs decreased by 2.5-fold showing that peptide binding site is located at or near the loop region containing the pseudouridines at positions 1911 and 1915. In addition, the effect of pH on the complex formation of H69 and UUU RNA with NQVANHQ-NH2 peptide was studied at two different pH values of 7.0 and 5.2. There was three-fold decrease of the apparent dissociation constant for the 1:1 complex of RNA and the peptide indicating that either protonation of the RNA or the peptide structure influenced this change in binding of the two species. The specificity of the peptide for H69 was tested with related RNA such as human H69 and unrelated RNAs such as helix 31 and A-site rRNA. The peptide showed three-fold lower affinity than the target H69 RNA for all these RNAs suggesting that the peptide has features for developing it as a lead compound for novel antimicrobial