A novel mechanism for the very first step in polypeptide elongation on the ribosome has recently been proposed by Simonson and Lake (2002). According to their transorientation hypothesis, the incoming aminoacyl-transfer RNA (aa-tRNA) initially binds to the messenger RNA (mRNA) with the anticodon stacked on the 5′ side of the anticodon loop; then, following GTP hydrolysis, the tRNA swings into the A-site by switching from the 5′-stacked conformation to the classic 3′-stacked geometry while maintaining Watson–Crick base pairing with the mRNA. There are several serious problems with this proposal.

Saccharomyces cerevisiae contains three nonessential genes (NGL1, NGL2, and NGL3) that encode proteins containing a domain with similarity to a Mg2+-dependent endonuclease motif present in the mRNA deadenylase Ccr4p. We have investigated a possible role of these proteins in rRNA processing, because for many of the pre-rRNA processing steps, the identity of the responsible nuclease remains elusive. Analysis of RNA isolated from cells in which the NGL2 gene has been inactivated (ngl2Δ) demonstrates that correct 3′-end formation of 5.8S rRNA at site E is strictly dependent on Ngl2p. No role in pre-rRNA processing could be assigned to Ngl1p and Ngl3p. The 3′-extended 5.8S rRNA formed in the ngl2Δ mutant is slightly shorter than the 6S precursor previously shown to accumulate upon combined deletion of the 3′ → 5′ exonuclease-encoding REX1 and REX2 genes or upon depletion of the exosomal subunits Rrp40p or Rrp45p. Thus, our data add a further component to the set of nucleases required for correct 3′-end formation of yeast 5.8S rRNA.

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II (pol II) plays an important role in promoting steps of pre-mRNA processing. To identify proteins in human cells that bind to the CTD and that could mediate its functions in pre-mRNA processing, we used the mouse CTD expressed in bacterial cells in affinity chromatography experiments. Two proteins present in HeLa cell extract, the splicing and transcription-associated factors, PSF and p54nrb/NonO, bound specifically and could be purified to virtual homogeneity by chromatography on immobilized CTD matrices. Both hypo- and hyperphosphorylated CTD matrices bound these proteins with similar selectivity. PSF and p54nrb/NonO also copurified with a holoenzyme form of pol II containing hypophosphorylated CTD and could be coimmunoprecipitated with antibodies specific for this and the hyperphosphorylated form of pol II. That PSF and p54nrb/NonO promoted the binding of RNA to immobilized CTD matrices suggested these proteins can interact with the CTD and RNA simultaneously. PSF and p54nrb/NonO may therefore provide a direct physical link between the pol II CTD and pre-mRNA processing components, at both the initiation and elongation phases of transcription.

Analysis of atomic resolution structures of the rRNAs within the context of the 50S and the 30S ribosomal subunits have revealed the presence of nine examples of a recurrent structural motif, first observed in the TΨC loop of tRNAs. The key component of this T-loop motif is a UA trans Watson–Crick/Hoogsteen base pair stacked on a Watson–Crick pair on one side. This motif is stabilized by several noncanonical hydrogen bonds, facilitating RNA–RNA as well as RNA–protein interactions. In particular, the sugar edge of the purine on the 3′ side of the pivotal uridine in the UA pair frequently forms a noncanonical base pair with a distant residue. The bulged-out bases, usually seen as part of the motif, also use their Watson–Crick edges to interact with nearby residues via base-specific hydrogen bonds. In certain occurrences, a backbone reversal is stabilized by specific hydrogen bonds as is observed in the U-turn motifs and the adenosine residue of the key UA pair interacts with a third base via its Watson–Crick edge, essentially generating a base triple.

Bacterial protein synthesis is the target for several classes of established antibiotics. This report describes the characterization of a novel translation inhibitor produced by the soil bacterium Flexibacter. The dipeptide antibiotic TAN1057 A/B was synthesized and designated GS7128. As reported previously, TAN1057 inhibits protein synthesis in both Escherichia coli and Staphylococcus aureus, leaving transcription unaffected. Cell-free translation systems from E. coli were used to further dissect the mechanism of translational inhibition. Binding of mRNA to ribosomes was unaffected by the drug, whereas the initiation reaction was reduced. Elongation of translation was completely inhibited by GS7128. Detailed analysis showed that the peptidyl transferase reaction was strongly inhibited, whereas tRNA binding to both A- and P-site was unaffected. Selection and analysis of drug-resistant mutants of S. aureus suggests that drug uptake may be mediated by a dipeptide transport mechanism.

Archaea-like bacteria are prokaryotes but, in contrast, use eukaryotic-like systems for key aspects of DNA, RNA, and protein metabolism. mRNA is typically unstable in bacteria and stable in eukaryotes, but little information is available about mRNA half-lives in archaea. Because archaea are generally insensitive to antibiotics, examination of mRNA stability in the hyperthermophile, Sulfolobus solfataricus, required the identification of transcription inhibitors for half-life determinations. An improved lacS promoter-dependent in vitro transcription system was used to assess inhibitor action. Efficient inhibitors were distinguished as blocking both lacSp transcription in vitro and the incorporation of 3H-uracil into bulk RNA in vivo. Actinomycin D was the most stable and potent compound identified. A survey of transcript chemical half-lives normalized to levels of the signal recognition particle 7S RNA ranged from at least 2 h for tfb1, a transcription factor TFIIB paralog, to a minimum of 6.3 min for gln1, one of three glutamine synthetase paralogs. Transcript half-lives for other mRNAs were: 2 h, superoxide dismutase (sod); 37.5 min, glucose dehydrogenase (dhg1); 25 min, alpha-glucosidase (malA); and 13.5 min, transcription factor TFIIB-2 (tfb2) resulting in a minimum average half-life of 54 min. These are the first mRNA half-lives reported for a hyperthermophile or member of the crenarchaea. The unexpected stability of several transcripts has important implications for gene expression and mRNA degradation in this organism.

There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis—the highly conserved 3′ end of 16S rRNA (aSD) complementary to the Shine–Dalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving β-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the β-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5′ untranslated region, it can ensure a substantial enhancing effect on translation.

Several cap-binding proteins from both the nucleus and cytosol have been identified that mediate processes such as pre-mRNA splicing, translation initiation, and mRNA turnover. Here we describe a novel cap-binding protein, pokeweed antiviral protein (PAP), a 29-kDa type I ribosome-inactivating protein (RIP) isolated from Phytolacca americana. In addition to depurinating the sarcin/ricin loop of the large rRNA, an activity common to all RIPs, we have reported recently that PAP depurinates capped, but not uncapped RNAs in vitro. Here we characterize this activity further and, using affinity chromatography, show that PAP binds to the m7Gppp cap structure. PAP UV-crosslinks to m7GpppG-capped luciferase mRNA more efficiently than GpppG-capped luciferase mRNA, indicating specificity for the methylated guanosine. We present evidence that PAP does not remove the cap structure or depurinate the m7Gppp as shown by primer extension of capped and uncapped luciferase transcripts incubated with PAP. Modeling studies of cap interaction with PAP predict that the cap structure would bind to the active site of PAP in a similar manner to guanine. We map the depurination sites on the capped luciferase RNA and illustrate that depurination occurs at specific adenine and guanine residues throughout the RNA sequence. Incubation of isolated ribosomes with PAP and increasing molar concentrations of m7GpppG relative to PAP resulted in a decrease in the level of rRNA depurination. Therefore, at elevated concentrations, the methylated cap structure competes with the adenine or guanine for binding to PAP, even though the affinity of PAP for capped message is almost fourfold lower than for rRNA. These results demonstrate that the activity of PAP is not limited to rRNA depurination, but that PAP binds to the cap structure and depurinates mRNAs downstream of the cap in vitro. These findings may have implications for understanding PAP activity in vivo.

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA–protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the αCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to αCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for αCP decoy action. Because αCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of αCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.

RNAs in the mitochondrion of Physarum polycephalum are edited by the precise cotranscriptional addition of nonencoded nucleotides. Here we describe experiments to address the basis of editing specificity using a series of chimeric templates generated by either rearranging the DNA present in editing-competent mitochondrial transcription elongation complexes (mtTECs) or linking it to exogenous DNA. Notably, run-on transcripts synthesized from rearranged mtTECs are edited at the natural sites, even when different genes are ligated together, yet exogenous, deproteinized DNA does not support editing. Furthermore, the accuracy of nucleotide insertion in chimeric RNAs argues that any cis-acting determinants of cytidine insertion are limited to small regions surrounding editing sites. Taken together, these observations strongly suggest that template-associated factors affect read-out of the mitochondrial genome.