Sequence and Properties of Cagein,a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as “basal-body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.     

Programmed translational frameshifting is likely required for expressions of genes encoding putative nuclear protein kinases of the ciliate Euplotes octocarinatus

Three macronuclear genes encoding putative nuclear protein kinases of the ciliate Euplotes octocarinatus syngen 1 were isolated and sequenced. All three deduced gene products share significant properties with a group of recently identified nuclear serine/threonine protein kinases named Ndr. The three predicted proteins contain the twelve conserved catalytic subdomains of protein kinases and 22 near universally-conserved amino acids residues that are characteristic of serine/threonine protein kinases. In addition, there is an approximately 30 amino acid-peptide insertion between subdomains VII and VIII that contains a potential nuclear localization signal. Sequence analysis suggests that expression of the Eondr2 gene requires a + 1 programmed translational frameshift for its translation. Comparison of the deduced EoNdr2 with other known Ndr protein kinases implies that a + 1 ribosomal frameshift occurs at the motif AAATAA.     

Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe)

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.     

Asc1, homolog of human RACK1, prevents frameshifting in yeast by ribosomes stalled at CGA codon repeats

Quality control systems monitor and stop translation at some ribosomal stalls, but it is unknown if halting translation at such stalls actually prevents synthesis of abnormal polypeptides. In yeast, ribosome stalling occurs at Arg CGA codon repeats, with even two consecutive CGA codons able to reduce translation by up to 50%. The conserved eukaryotic Asc1 protein limits translation through internal Arg CGA codon repeats. We show that, in the absence of Asc1 protein, ribosomes continue translating at CGA codons, but undergo substantial frameshifting with dramatically higher levels of frameshifting occurring with additional repeats of CGA codons. Frameshifting depends upon the slow or inefficient decoding of these codons, since frameshifting is suppressed by increased expression of the native tRNA^Arg(ICG) that decodes CGA codons by wobble decoding. Moreover, the extent of frameshifting is modulated by the position of the CGA codon repeat relative to the translation start site. Thus, translation fidelity depends upon Asc1-mediated quality control.     

Prophage lambda at unusual chromosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12

An Escherichia coli strain deleted for the primary λ attachment site was lysogenized with λ at secondary sites. Some lysogens became mutants because of prophage insertion in the affected gene. Mutagenesis by phage λ is not random with respect to the gene affected: most mutants were pro, although certain other genes could be mutated at lower frequencies. In the case of several independent ilv and gal mutants, the sites of prophage insertion were in the same segment of the ilv region and galT gene respectively. The galT location may also be a preferred site for the insertion of DNAs other than prophage λ. Insertion of prophage λ within an operon can reduce the expression of operator-distal genes. A trpC λ insertion mutant expresses the operator-distal trpB function constitutively at a low level. This expression probably derives from a promoter located in the left arm of the prophage.     

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.     

Frequent appearance of novel protein-coding sequences by frameshift translation

Genomic duplication, followed by divergence, contributes to organismal evolution. Several mechanisms, such as exon shuffling and alternative splicing, are responsible for novel gene functions, but they generate homologous domains and do not usually lead to drastic innovation. Major novelties can potentially be introduced by frameshift mutations and this idea can explain the creation of novel proteins. Here, we employ a strategy using simulated protein sequences and identify 470 human and 108 mouse frameshift events that originate new gene segments. No obvious interspecies overlap was observed, suggesting high rates of acquisition of evolutionary events. This inference is supported by a deficiency of TpA dinucleotides in the protein-coding sequences, which decreases the occurrence of translational termination, even on the complementary strand. Increased usage of the TGA codon as the termination signal in newer genes also supports our inference. This suggests that tolerated frameshift changes are a prevalent mechanism for the rapid emergence of new genes and that protein-coding sequences can be derived from existing or ancestral exons rather than from events that result in noncoding sequences becoming exons.     

Stepwise Evolution and Exceptional Conservation of ORF1a/b Overlap in Coronaviruses

The programmed frameshift element (PFE) rerouting translation from ORF1a to ORF1b is essential for the propagation of coronaviruses. The combination of genomic features that make up PFE—the overlap between the two reading frames, a slippery sequence, as well as an ensemble of complex secondary structure elements—places severe constraints on this region as most possible nucleotide substitution may disrupt one or more of these elements. The vast amount of SARS-CoV-2 sequencing data generated within the past year provides an opportunity to assess the evolutionary dynamics of PFE in great detail. Here, we performed a comparative analysis of all available coronaviral genomic data available to date. We show that the overlap between ORF1a and ORF1b evolved as a set of discrete 7, 16, 22, 25, and 31 nucleotide stretches with a well-defined phylogenetic specificity. We further examined sequencing data from over 1,500,000 complete genomes and 55,000 raw read data sets to demonstrate exceptional conservation and detect signatures of selection within the PFE region.