Internal Reading Frame Research Articles

Dual Toxic-Peptide-Coding Staphylococcus aureus RNA under Antisense Regulation Targets Host Cells and Bacterial Rivals Unequally

Produced from the pathogenicity islands of Staphylococcus aureus clinical isolates, stable SprG1 RNA encodes two peptides from a single internal reading frame. These two peptides accumulate at the membrane, and inducing their expression triggers S. aureus death. Replacement of the two initiation codons by termination signals reverses this toxicity. During growth, cis-antisense RNA SprF1 is expressed, preventing mortality by reducing SprG1 RNA and peptide levels. The peptides are secreted extracellularly, where they lyse human host erythrocytes, a process performed more efficiently by the longer peptide. The two peptides also inactivate Gram-negative and -positive bacteria, with the shorter peptide more effective against S. aureus rivals. Two peptides are secreted from an individual RNA containing two functional initiation codons. Thus, we present an unconventional type I toxin-antitoxin system expressed from a human pathogen producing two hemolytic and antibacterial peptides from a dual-coding RNA, negatively regulated by a dual-acting antisense RNA.

Small protein B interacts with the large and the small subunits of a stalled ribosome during trans-translation

During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10−10 M, similar to its KD for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes.

Induction of Prothrombinase fgl2 by the Nucleocapsid Protein of Virulent Mouse Hepatitis Virus Is Dependent on Host Hepatic Nuclear Factor-4α

Fibrinogen-like protein 2/fibroleukin (Fgl2) plays a pivotal role in the pathogenesis of both experimental and human fulminant hepatic failure. We have reported recently that the nucleocapsid (N) protein from strains of murine hepatitis virus (MHV-3, MHV-A59), which cause massive hepatocellular necrosis but not from strains (MHV-JHM, MHV-2) which do not produce serious liver disease, induces transcription of fgl2. The purpose of the present study was to characterize both viral and host factor(s) necessary for viral induced transcription of fgl2. Mutation of residues Gly-12, Pro-38, Asn-40, Gln-41, and Asn-42 within domain 1 of the N protein of MHV-A59 to their corresponding residues found in MHV-2 abrogated fgl2 transcription, whereas mutation of other N protein domains, including a protein expressed from an internal reading frame (I protein), did not affect fgl2 gene transcription. We then examined the -372 to -306 sequence within the 1.3-kb fgl2 promoter region upstream from the transcription start site that was previously identified as necessary for N protein-induced gene transcription. We demonstrated that the -331/-325 HNF4 cis-element and its cognate transcription factor, HNF4alpha, are necessary for virus-induced fgl2 gene transcription. In uninfected macrophages and macrophages infected with MHV-2, an unidentified protein occupies the HNF4 cis-element. Following stimulation with MHV-A59, it was shown by electrophoretic mobility shift assay that HNF4alpha binds the HNF4 cis-element in the fgl2 promoter. We further report the unprecedented presence of HNF4alpha in peritoneal macrophages. Collectively, the results of this study define both viral and host factors necessary for induction of fgl2 prothrombinase gene transcription in MHV infection and may provide an explanation for the hepatotrophic nature of MHV-induced fulminant hepatic failure.

Construction of Ovine Adenovirus Recombinants by Gene Insertion or Deletion of Related Terminal Region Sequences

An ovine adenovirus which may be the prototype for a new group of adenoviruses has been engineered as a gene transfer vector. One recombinant containing a 0.95-kb insertion expressed a sheep parasite antigen from the ovine adenovirus major late promoter and tripartite leader sequences. It was shown that insertions of at least 4.3 kb were tolerated at either one of two sites in the genome without the introduction of a compensating deletion. The unique structure of this viral genome was further emphasized by the discovery that four open reading frames at the right hand end show significant identity to each other but not to other sequences in the databases. Two other unrelated open reading frames were also present. RT-PCR analysis identified two transcripts in this region which were derived from a promoter which was located very close to, or within the ITR sequence. Splicing removed all but the first and last of the ORFs from these RNAs, suggesting that some sequences might be nonessential for replicationin vitro.A ∼2-kb deletion, which removed or truncated the internal reading frames was introduced into the region without affecting virus viability. The carrying capacity of OAV recombinants should therefore be at least 6.3 kb. The relative packaging capacity of OAV (114%) therefore exceeds that of Ad5 (105%), although a comparison of virus particle sizes by electron microscopy showed that OAV was smaller than Ad5. These studies improve the potential utility of OAV as a gene transfer vector.

Evidence for a group II intron in Escherichia coli inserted into a highly conserved reading frame associated with mobile DNA sequences.

The distribution of group II introns in the living world is an important aspect of the hypothesis which postulates their evolutionary relation to the nuclear spliceosome. As an alternative to the restricted experimental approaches towards their identification we devised a strategy to recognize group II introns in sequence data. By this approach we identified a locus on a plasmid in the bacterium Escherichia coli. Modelling of the derived RNA secondary structure reveals the presence of perfectly conserved domains V and VI as typical features of group II introns. An intron internal reading frame upstream of domain V is homologous to group II intron encoded maturases. A reading frame downstream of the predicted 3'-splice site is highly similar to a small polypeptide encoded in the central part of the Agrobacterium tumefaciens T-DNA. With the TBLASTN algorithm a set of plasmid-borne insertion sequences in Agrobacteria and Rhizobia and surprisingly also in a Yersinia pseudotuberculosis strain was identified which contain this highly conserved reading frame.

Plant retrotransposon from Lilium henryi is related to Ty3 of yeast and the gypsy group of Drosophila.

The lily retrotransposon del 1-46 is 9345 base pairs (bp) long. It has long terminal repeats (LTRs) of 2406 bp (left) and 2415 bp (right), which differ in sequence by 1.4%. Sequences similar to those involved in priming DNA synthesis in retroviruses occur in the internal region. Near the left LTR is a sequence complementary to 18 residues at the 3' end of methionine initiator tRNA of three plant species, and a run of 12 purines occurs close to the right LTR. One internal reading frame of del 1-46 has relatively few stop codons. The 1462-codon product from this frame has motifs, in N to C terminus order, corresponding to those identified with RNA binding, protease, reverse transcriptase, RNase H, and integrase functions in retroviruses and certain other retrotransposons. Amino acid sequence comparisons of three conserved pol regions show del to be closely related to the Ty3 retrotransposon of yeast (37-40% identity). del is also related to the gypsy group of Drosophila (17.6, 297, gypsy/mdg4, and 412), showing closer identity with their reverse transcriptase (32-38%) and RNase H (36-45%) domains than with their integrase domain (21-26%). It is proposed that a gypsy group ancestor exchanged the integrase region with a more distantly related element since its divergence from a del/Ty3 common ancestor. The occurrence of related retrotransposons in three different kingdoms (plants, animals, and fungi) strongly implies their horizontal transmission in recent evolutionary time.

An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene

The intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae possesses a long internal reading frame (ORF) that is conserved in various yeast species. In crosses between intron-plus and intron-minus variants, this intron determines a specific gene conversion phenomenon, which results in the integration of the intron sequence within all previously intron-minus copies of the gene. We show, from a frameshift mutant within the intron ORF and from the need of mitochondrial protein synthesis, that ORF encodes a protein active in the gene conversion that spreads the intron within populations of interbreeding strains. This new intron function is reminiscent of the "transposase" encoded by mobile genetic elements and is discussed in relation to other intron functions.