Extensive Secondary Structure Research Articles

Protein phosphatase inhibitor-1 (PPI-1) has protective activities in stress conditions in E. coli

Protein phosphatase inhibitor-1 (PPI-1) is a major inhibitor of protein phosphatase 1 (PP1), which regulates signal transduction in many eukaryotic cellular processes. Biophysical studies have shown that PPI-1 has a large Stokes radius and is heat stable, suggesting that it lacks extensive secondary structures. The unfolded structure of PPI-1 may enable it to interact with many proteins or ligands during stress conditions. Here we show that PPI-1 can act as a protective molecule, inhibiting protein aggregation and guarding E. coli cells against various stresses. Therefore, PPI-1 seems to have a physiological function as a protective molecule as well as regulator of protein serine/threonine phosphatases.

Chemical synthesis and structure elucidation of bovine κ-casein (1–44)

The caseins (α s1, α s2, β, and κ) are phosphoproteins present in bovine milk that have been studied for over a century and whose structures remain obscure. Here we describe the chemical synthesis and structure elucidation of the N-terminal segment (1–44) of bovine κ-casein, the protein which maintains the micellar structure of the caseins. κ-Casein (1–44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nuclear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited considerable helical structure. Further, NMR analysis showed the presence of a helical segment containing 26 residues which extends from Pro 8 to Arg 34. This is the first report which demonstrates extensive secondary structure within the casein class of proteins.

The Maize An2 Gene is Induced by Fusarium Attack and Encodes an ent-Copalyl Diphosphate Synthase

Using the technique of differential display, a maize transcript was identified whose silk tissue expression is induced in the presence of the ear rot pathogen Fusarium graminearum. The 3445 nt transcript includes a 727 nt 5' untranslated leader with the potential for extensive secondary structure and represents the maize gene An2. An2 encodes a copalyl diphosphate synthase (CPS)-like protein with 60% amino acid sequence identity with the maize An1 gene product involved in gibberellin (GA) biosynthesis. Recombinant expression and functional analysis demonstrated that both AN1 and AN2 are ent-copalyl diphosphate (ent-CPP) synthases (ent-CPS). Notably, the presence of an additional ent-CPS gene is consistent with previous reports that maize GA biosynthesis can proceed in the absence of An1. In addition, northern blot analysis showed that An2 transcript levels were strongly up-regulated by Fusarium attack, with an increase in silk, husk and ear tip tissues as early as 6 h after inoculation of silk channels with spore suspensions of various Fusarium sp. Gene expression of a third maize CPS-like gene, Cpsl1, is not affected by Fusarium infection. The Fusarium-inducible nature of An2 is also consistent with a previous report that cell-free extracts from maize seedlings produce ent-CPP derived diterpenes in response to Fusarium infection. However, it is not known whether An2 is involved in defense-related secondary metabolism in addition to GA synthesis.

Elevation of RNase R in Response to Multiple Stress Conditions

Cells respond to adverse environmental conditions by synthesizing new proteins or elevating the levels of pre-existing ones that are needed to cope with the particular stress situation. We show here that Escherichia coli RNase R, a processive 3'-to5'-exoribonuclease, is dramatically increased in response to a variety of different stress conditions. Elevation of RNase R activity by as much as 10-fold was observed in response to entry into stationary phase, starvation, and cold shock, and a approximately 3-fold increase was seen during growth in minimal medium compared with rich medium. The elevation in RNase R activity was associated primarily with an increase in RNase R protein. RNase R was previously implicated in quality control of rRNA and tRNA and in the decay of mRNAs with extensive secondary structure. Its dramatic increase under multiple stress conditions suggests extensive remodeling of structured RNA in response to the altered environment.

Gir2 is an intrinsically unstructured protein that is present in Saccharomyces cerevisiae as a group of heterogeneously electrophoretic migrating forms

Gir2 is a highly acidic cytoplasmic protein of Saccharomyces cerevisiae of unknown function that shows an anomalous migration on SDS–PAGE. Based on its large Stokes radius and thermostability, we have previously suggested that Gir2 lacks extensive secondary structure. Here we report that Gir2 is extremely sensitive to proteolysis when compared to glutathione- S-transferase, a highly structured protein, further indicating its unfolded nature. Prediction based on the FoldIndex program also indicates that Gir2 is a disordered protein. Using truncated forms of Gir2 we show that the N-terminal half of this protein, with its high content of acidic amino acid residues, is responsible for the anomalous electrophoretic behavior of Gir2. Because all these features are hallmarks of intrinsically unstructured proteins (IUP), we propose that Gir2 is another representative of the IUP group of proteins. Additionally, we describe that the endogenous yeast Gir2 shows heterogeneous electrophoretic mobility, which is not due to proteolytic cleavage.

Analysis of subsecond protein dynamics by amide hydrogen exchange and mass spectrometry using a quenched‐flow setup

Amide hydrogen exchange (HX) in combination with mass spectrometry (MS) is a powerful tool to analyze the folding and dynamics of proteins. In the traditional methodology the exchange time is controlled by manual pipetting, thereby limiting the time resolution to several seconds. Some conformational changes in proteins, however, occur in the subsecond time scale, making it desirable to perform HX at shorter time intervals down to the limit set by the intrinsic chemical exchange rate. We now report the development of the first completely on-line quenched-flow setup that allows the performance of HX experiments in the 100-sec to 30-sec time scale, on-line proteolytic digestion using immobilized proteases, rapid desalting, and MS analysis. We show that conformational fluctuations in the range of seconds can be detected and protection factors as small as 10 reproducibly determined. Using this setup we investigated the conformational properties of Escherichia coli heat-shock transcription factor sigma32 free in solution. Our results indicate that the C-terminal sigma4 domain of sigma32, which is responsible for the recognition of the -35 region of heat shock promoters, contains more extensive secondary structure than expected when compared with the structure of the homologous sigma-factor sigmaA in complex with the RNA-polymerase. This setup should be very useful for a more accurate analysis of structural motions in proteins in the subsecond to second time scale relevant to allostery and enzyme function.

An Important Role for RNase R in mRNA Decay

mRNA decay is a major determinant of gene expression. In Escherichia coli, message degradation initiates with an endoribonucleolytic cleavage followed by exoribonuclease digestion to generate 5′-mononucleotides. Although the 3′ to 5′ processive exoribonucleases, PNPase and RNase II, have long been considered to be mediators of this digestion, we show here that another enzyme, RNase R, also participates in the process. RNase R is particularly important for removing mRNA fragments with extensive secondary structure, such as those derived from the many mRNAs that contain REP elements. In the absence of RNase R and PNPase, REP-containing fragments accumulate to high levels. RNase R is unusual among exoribonucleases in that, by itself, it can digest through extensive secondary structure provided that a single-stranded binding region, such as a poly(A) tail, is present. These data demonstrate that RNase R, which is widespread in prokaryotes and eukaryotes, is an important participant in mRNA decay.

Conservation and divergence of microRNA families in plants

Background MicroRNAs (miRNAs) are 20 to 24 nucleotides short RNAs involved in posttranscriptional regulation in plants and animals. MiRNAs are processed from larger precursors with extensive secondary structure. In plants, a total of 286 miRNA genes in Arabidopsis, rice and maize had been identified by March 2005, clustered in 43 families.

Translational Regulation of Hsp90 mRNA: AUG-PROXIMAL 5′-UNTRANSLATED REGION ELEMENTS ESSENTIAL FOR PREFERENTIAL HEAT SHOCK TRANSLATION

Heat shock in Drosophila results in repression of most normal (non-heat shock) mRNA translation and the preferential translation of the heat shock mRNAs. The sequence elements that confer preferential translation have been localized to the 5'-untranslated region (5'-UTR) for Hsp22 and Hsp70 mRNAs (in Drosophila). Hsp90 mRNA is unique among the heat shock mRNAs in having extensive secondary structure in its 5'-UTR and being abundantly represented in the non-heat shocked cell. In this study, we show that Hsp90 mRNA translation is inefficient at normal growth temperature, and substantially activated by heat shock. Its preferential translation is not based on an IRES-mediated translation pathway, because overexpression of eIF4E-BP inhibits its translation (and the translation of Hsp70 mRNA). The ability of Hsp90 mRNA to be preferentially translated is conferred by its 5'-UTR, but, in contrast to Hsp22 and -70, is primarily influenced by nucleotides close to the AUG initiation codon. We present a model to account for Hsp90 mRNA translation, incorporating results indicating that heat shock inhibits eIF4F activity, and that Hsp90 mRNA translation is sensitive to eIF4F inactivation.

Detailed mapping of RNA secondary structures in core and NS5B-encoding region sequences of hepatitis C virus by RNase cleavage and novel bioinformatic prediction methods.

There is accumulating evidence from bioinformatic studies that hepatitis C virus (HCV) possesses extensive RNA secondary structure in the core and NS5B-encoding regions of the genome. Recent functional studies have defined one such stem-loop structure in the NS5B region as an essential cis-acting replication element (CRE). A program was developed (STRUCTUR_DIST) that analyses multiple rna-folding patterns predicted by mfold to determine the evolutionary conservation of predicted stem-loop structures and, by a new method, to analyse frequencies of covariant sites in predicted RNA folding between HCV genotypes. These novel bioinformatic methods have been combined with enzymic mapping of RNA transcripts from the core and NS5B regions to precisely delineate the RNA structures that are present in these genomic regions. Together, these methods predict the existence of multiple, often juxtaposed stem-loops that are found in all HCV genotypes throughout both regions, as well as several strikingly conserved single-stranded regions, one of which coincides with a region of the genome to which ribosomal access is required for translation initiation. Despite the existence of marked sequence conservation between genotypes in the HCV CRE and single-stranded regions, there was no evidence for comparable suppression of variability at either synonymous or non-synonymous sites in the other predicted stem-loop structures. The configuration and genetic variability of many of these other NS5B and core structures is perhaps more consistent with their involvement in genome-scale ordered RNA structure, a structural configuration of the genomes of many positive-stranded RNA viruses that is associated with host persistence.

Stable and noncompetitive RNA internal control for routine clinical diagnostic reverse transcription-PCR.

Clinical diagnostic tests based on nucleic acid amplification assist with the prompt diagnosis of microbial infections because of their speeds and extremely low limits of detection. However, the design of appropriate internal controls for such assays has proven difficult. We describe a reaction-specific RNA internal control for diagnostic reverse transcription (RT)-PCR which allows extraction, RT, amplification, and detection to be monitored. The control consists of a G+C-rich (60%) RNA molecule with an extensive secondary structure, based on a modified hepatitis delta virus genome. The rod-like structure of this RNA, with 70% intramolecular base pairing, provides a difficult template for RT-PCR. This ensures that the more favorable target virus amplicon is generated in preference to the control, with the control being detected only if the target virus is absent. The unusual structure of hepatitis delta virus RNA has previously been shown to enhance its stability and resistance to nucleases, an advantage for routine use as an internal control. The control was implemented in three nested multiplex RT-PCRs to detect nine clinically important respiratory viruses: (i) influenza A and B viruses, (ii) respiratory syncytial viruses A and B and human metapneumovirus, and (iii) parainfluenza virus types 1 to 4. The detection limits of these assays were not detectably compromised by the presence of the RNA control. During routine testing of 324 consecutive unselected respiratory samples, the presence of the internal control ensured that genuine and false-negative results were distinguishable, thus increasing the diagnostic confidence in the assay.

The Denatured State of Engrailed Homeodomain under Denaturing and Native Conditions

Protein folding starts from the elusive form of the denatured state that is present under conditions that favour the native state. We have studied the denatured state of Engrailed Homeodomain (En-HD) under mildly and strongly denaturing conditions at the level of individual residues by NMR and more globally by conventional spectroscopy and solution X-ray scattering. We have compared these states with a destabilized mutant, L16A, which is predominantly denatured under conditions where the wild-type is native. This engineered denatured state, which could be directly studied under native conditions, was in genuine equilibrium with the native state, which could be observably populated by changing the conditions or introducing a stabilizing mutation. The denatured state had extensive native secondary structure and was significantly compact and globular. But, the side-chains and backbone were highly mobile. Non-cooperative melting of the residual structure on the denatured state of En-HD was observed, both at the residue and the molecular level, with increasingly denaturing conditions. The absence of a co-operative transition could result from the denatured state ensemble progressing through a series of intermediates or from a more general slide (second-order transition) from the compact form under native conditions to the more extended at highly denaturing conditions. In either case, the starting point for folding under native conditions is highly structured and already poised to adopt the native structure.

Dimerization and template switching in the 5' untranslated region between various subtypes of human immunodeficiency virus type 1.

The human immunodeficiency virus type 1 (HIV-1) particle contains two identical RNA strands, each corresponding to the entire genome. The 5' untranslated region (UTR) of each RNA strand contains extensive secondary and tertiary structures that are instrumental in different steps of the viral replication cycle. We have characterized the 5' UTRs of nine different HIV-1 isolates representing subtypes A through G and, by comparing their homodimerization and heterodimerization potentials, found that complementarity between the palindromic sequences in the dimerization initiation site (DIS) hairpins is necessary and sufficient for in vitro dimerization of two subtype RNAs. The 5' UTR sequences were used to design donor and acceptor templates for a coupled in vitro dimerization-reverse transcription assay. We showed that template switching during reverse transcription is increased with a matching DIS palindrome and further stimulated proportional to the level of homology between the templates. The presence of the HIV-1 nucleocapsid protein NCp7 increased the template-switching efficiency for matching DIS palindromes twofold, whereas the recombination efficiency was increased sevenfold with a nonmatching palindrome. Since NCp7 did not effect the dimerization of nonmatching palindromes, we concluded that the protein most likely stimulates the strand transfer reaction. An analysis of the distribution of template-switching events revealed that it occurs throughout the 5' UTR. Together, these results demonstrate that the template switching of HIV-1 reverse transcriptase occurs frequently in vitro and that this process is facilitated mainly by template proximity and the level of homology.

Replacement of murine leukemia virus readthrough mechanism by human immunodeficiency virus frameshift allows synthesis of viral proteins and virus replication.

Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model.

The mRNA of the translationally controlled tumor protein P23/TCTP is a highly structured RNA, which activates the dsRNA-dependent protein kinase PKR.

The dsRNA-activated protein kinase PKR is involved in signal transduction pathways that mediate cellular processes as diverse as cell growth and differentiation, the stress response, and apoptosis. PKR was originally described as an interferon-inducible elF2alpha kinase involved in the antiviral defense mechanism of the cell. The interaction of the kinase with specific viral RNAs has been studied in much detail, but information about cellular mRNAs, which are able to bind and activate PKR, is scarce. In search for such cellular mRNAs, we developed a cloning strategy to identify individual mRNA species from the dsRNA-rich fraction of Daudi cell poly(A)+ RNA. Two out of five cDNA clones we obtained contained sequences derived from the mRNA of the translationally controlled tumor protein P23/TCTP, indicating that this mRNA is present in the dsRNA-rich fraction. Secondary structure predictions and gel electrophoretic mobility investigations on P23/TCTP transcripts confirmed the potential of this mRNA to form extensive secondary structure. A full-length P23 transcript, but not a truncated version thereof, was able to bind to PKR in vitro and in vivo. Transient transfection experiments in human 293 cells showed that coexpression of full-length P23 mRNA leads to partial inhibition of the expression of a beta-galactosidase reporter gene in trans. Additional coexpression of a dominant negative mutant of PKR or of adenovirus VA1 RNA suppressed this inhibition, indicating that it is mediated by PKR. Studies on P23/TCTP expression in cells from PKR-knockout mice suggest that P23/TCTP mRNA translation is regulated by PKR. Hence, our results demonstrate that the mRNA of P23/TCTP may both activate PKR and be subject to translational regulation by this kinase.

Evolutionarily conserved RNA secondary structures in coding and non-coding sequences at the 3' end of the hepatitis G virus/GB-virus C genome.

Hepatitis G virus (HGV)/GB virus C (GBV-C) causes persistent, non-pathogenic infection in a large proportion of the human population. Epidemiological and genetic evidence indicates a long-term association between HGV/GBV-C and related viruses and a range of primate species, and the co-speciation of these viruses with their hosts during primate evolution. Using a combination of covariance scanning and analysis of variability at synonymous sites, we previously demonstrated that the coding regions of HGV/GBV-C may contain extensive secondary structure of undefined function (Simmonds & Smith, Journal of Virology 73, 5787-5794, 1999 ). In this study we have carried out a detailed comparison of the structure of the 3'untranslated region (3'UTR) of HGV/GBV-C with that of the upstream NS5B coding sequence. By investigation of free energies on folding, secondary structure predictive algorithms and analysis of covariance between HGV/GBV-C genotypes 1-4 and the more distantly related HGV/GBV-C chimpanzee variant, we obtained evidence for extensive RNA secondary structure formation in both regions. In particular, the NS5B region contained long stem-loop structures of up to 38 internally paired nucleotides which were evolutionarily conserved between human and chimpanzee HGV/GBV-C variants. The prediction of similar structures in the same region of hepatitis C virus may allow the functions of these structures to be determined with a more tractable experimental model.

Selective binding by the RNA binding domain of PKR revealed by affinity cleavage.

The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.

The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region.

Human endogenous retrovirus K (HERV-K) is the name given to an approximately 30-million-year-old family of endogenous retroviruses present at >50 copies per haploid human genome. Previously, the HERV-K were shown to encode a nuclear RNA export factor, termed K-Rev, that is the functional equivalent of the H-Rev protein encoded by human immunodeficiency virus type 1. HERV-K was also shown to contain a cis-acting target element, the HERV-K Rev response element (K-RRE), that allowed the nuclear export of linked RNA transcripts in the presence of either K-Rev or H-Rev. Here, we demonstrate that the functionally defined K-RRE coincides with a statistically highly significant unusual RNA folding region and present a potential RNA secondary structure for the approximately 416-nt K-RRE. Both in vitro and in vivo assays of sequence specific RNA binding were used to map two primary binding sites for K-Rev, and one primary binding site for H-Rev, within the K-RRE. Of note, all three binding sites map to discrete predicted RNA stem-loop subdomains within the larger K-RRE structure. Although almost the entire 416-nt K-RRE was required for the activation of nuclear RNA export in cells expressing K-Rev, mutational inactivation of the binding sites for K-Rev resulted in the selective loss of the K-RRE response to K-Rev but not to H-Rev. Together, these data strongly suggest that the K-RRE, like the H-RRE, coincides with an extensive RNA secondary structure and identify specific sites within the K-RRE that can recruit either K-Rev or H-Rev to HERV-K RNA transcripts.

Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.)

Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

Mutational Analysis of the 5′ Leader Region of Simian Foamy Virus Type 1

The sequence within the 5′ untranslated region of the retroviral genome contains important cis elements for many steps in viral replication. There is limited information available on the role of this region in foamy virus replication. Similar to other retroviruses, the 5′ untranslated region of foamy viruses predicts extensive RNA secondary structure. Serial mutations that could change parts of the predicted secondary structure were introduced in the 5′ leader sequence including the R-U5 region of simian foamy virus type 1 (SFV-1) to investigate their role in virus genome packaging and virus replication. Point mutations in the R-U5 regions at nucleotide positions 7–12 (I), 241–243 (B), and 256–257 (D) had no effect on virus replication. Base substitution mutation at positions 193–195 (C), however, severely impaired virus replication. Deletion of sequences in the leader region, between the primer-binding site and the gag gene, at positions 364–399 (d1), 397–435 (d2), or 364–435 (d3), which included sequences for RNA genome dimerization, also blocked SFV-1 replication. Interestingly, none of these mutations affected genome packaging or the synthesis of viral transcripts, suggesting that a step(s) of virus replication following packaging is affected. The region between the primer-binding site and the gag gene, therefore, is not essential for foamy virus genome packaging. Furthermore, the cis-acting elements for genome dimerization and packaging appear to be localized in separate regions for foamy viruses.