Local Structural and Environmental Factors Define the Efficiency of an RNA Pseudoknot Involved in Programmed Ribosomal Frameshift Process

In programmed -1 ribosomal frameshift, an RNA pseudoknot stalls the ribosome at specific sequence and restarts translation in a new reading frame. A precise understanding of structural characteristics of these pseudoknots and their PRF inducing ability has not been clear to date. To investigate this phenomenon, we have studied various structural aspects of a -1 PRF inducing RNA pseudoknot from BWYV using extensive molecular dynamics simulations. A set of functional and poorly functional forms, for which previous mutational data were available, were chosen for analysis. These structures differ from each other by either single base substitutions or base-pair replacements from the native structure. We have rationalized how certain mutations in RNA pseudoknot affect its function; e.g., a specific base substitution in loop 2 stabilizes the junction geometry by forming multiple noncanonical hydrogen bonds, leading to a highly rigid structure that could effectively resist ribosome-induced unfolding, thereby increasing efficiency. While, a CG to AU pair substitution in stem 1 leads to loss of noncanonical hydrogen bonds between stems and loop, resulting in a less stable structure and reduced PRF inducing ability, inversion of a pair in stem 2 alters specific base-pair geometry that might be required in ribosomal recognition of nucleobase groups, negatively affecting pseudoknot functioning. These observations illustrate that the ability of an RNA pseudoknot to induce -1 PRF with an optimal rate depends on several independent factors that contribute to either the local conformational variability or geometry

Specific mutations in a viral RNA pseudoknot drastically change ribosomal frameshifting efficiency

Many viruses regulate protein synthesis by −1 ribosomal frameshifting using an RNA pseudoknot. Frameshifting is vital for viral reproduction. Using the information gained from the recent high-resolution crystal structure of the beet western yellow virus pseudoknot, a systematic mutational analysis has been carried out in vitro and in vivo. We find that specific nucleotide tertiary interactions at the junction between the two stems of the pseudoknot are crucial. A triplex is found between stem 1 and loop 2, and triplex interactions are required for frameshifting function. For some mutations, loss of one hydrogen bond is sufficient to abolish frameshifting. Furthermore, mutations near the 5′ end of the pseudoknot can increase frameshifting by nearly 300%, possibly by modifying ribosomal contacts. It is likely that the selection of suitable mutations can thus allow viruses to adjust frameshifting efficiencies and thereby regulate protein synthesis in response to environmental change.

Differential effects on the hepatitis C virus (HCV) internal ribosome entry site by vitamin B-12 and the HCV core protein

To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical swine fever virus IRES elements. This permitted an examination of the role of domain IV in the control of HCV translation. A specific inhibitor of the HCV IRES, vitamin B-12 was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV and the GB virus B IRES elements, whereas the HCV core protein could only suppress translation from the wild-type HCV IRES. Thus, the mechanisms of translation inhibition by vitamin B-12 and the core protein differ, and they target different regions of the IRES.

A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain

An RNA molecule with properties of a satellite RNA was found in an isolate of barley yellow dwarf virus (BYDV), RPV serotype. It is 322 nucleotides long, single-stranded, and does not hybridize to the viral genome. Dimers of the RNA, which presumably represent replicative intermediates, were able to self-cleave into monomers. In vitro transcripts from cDNA clones were capable of self-cleavage in both the plus (encapsidated) and minus orientations. The sequence flanking the minus strand cleavage site contained a consensus " hammerhead" structure, similar to those found in other self-cleaving satellite RNAs. Although related to the hammerhead structure, sequences flanking the plus strand termini showed differences from the consensus and may be folded into a different structure containing a pseudoknot. © 1991.

Re-analysis of cryoEM data on HCV IRES bound to 40S subunit of human ribosome integrated with recent structural information suggests new contact regions between ribosomal proteins and HCV RNA

In this study, we combine available high resolution structural information on eukaryotic ribosomes with low resolution cryo-EM data on the Hepatitis C Viral RNA (IRES) human ribosome complex. Aided further by the prediction of RNA-protein interactions and restrained docking studies, we gain insights on their interaction at the residue level. We identified the components involved at the major and minor contact regions, and propose that there are energetically favorable local interactions between 40S ribosomal proteins and IRES domains. Domain II of the IRES interacts with ribosomal proteins S5 and S25 while the pseudoknot and the downstream domain IV region bind to ribosomal proteins S26, S28 and S5. We also provide support using UV cross-linking studies to validate our proposition of interaction between the S5 and IRES domains II and IV. We found that domain IIIe makes contact with the ribosomal protein S3a (S1e). Our model also suggests that the ribosomal protein S27 interacts with domain IIIc while S7 has a weak contact with a single base RNA bulge between junction IIIabc and IIId. The interacting residues are highly conserved among mammalian homologs while IRES RNA bases involved in contact do not show strict conservation. IRES RNA binding sites for S25 and S3a show the best conservation among related viral IRESs. The new contacts identified between ribosomal proteins and RNA are consistent with previous independent studies on RNA-binding properties of ribosomal proteins reported in literature, though information at the residue level is not available in previous studies.

RNA-Strukturen im Hepatitis-C-Virus und ihre Bedeutung für Translation und Replikation

Das Hepatitis C Virus (HCV) ist ein umhülltes Virus aus der Familie der Flaviviridae. Es besitzt ein Plusstrang-RNA Genom von ca. 9600 Nukleotiden Länge, das nur ein kodierendes Leseraster besitzt. Das Genom wird am 5’ und 3’ Ende von nicht-translatierten Sequenzen (NTRs) flankiert, welche für die Translation und vermutlich auch Replikation von Bedeutung sind. Die 5’ NTR besitzt eine interne Ribosomeneintrittsstelle (IRES), die eine cap-unabhängige Translation des ca. 3000 Aminosäure langen viralen Polyproteins erlaubt. Dieses wird ko- und posttranslational von zellulären und viralen Proteasen in 10 funktionelle Komponenten gespalten. Inwieweit die 5’ NTR auch für die Replikation der HCV RNA benötigt wird, war zu Beginn der Arbeit nicht bekannt. Die 3’ NTR besitzt eine dreigeteilte Struktur, bestehend aus einer variablen Region, dem polyU/UC-Bereich und der sogenannten X-Sequenz, eine hochkonservierte 98 Nukleotide lange Region, die vermutlich für die RNA-Replikation und möglicherweise auch für die Translation benötigt wird. Die genuae Rolle der 3’ NTR für diese beiden Prozesse war zu Beginn der Arbeit jedoch nicht bekannt. Ziel der Dissertation war deshalb eine detaillierte genetische Untersuchung der NTRs hinsichtlich ihrer Bedeutung für die RNA-Translation und -Replikation. In die Analyse mit einbezogen wurden auch RNA-Strukturen innerhalb der kodierenden Region, die zwischen verschiedenen HCV-Genotypen hoch konserviert sind und die mit verschiedenen computer-basierten Modellen vorhergesagt wurden. Zur Kartierung der für RNA-Replikation benötigten Minimallänge der 5’ NTR wurde eine Reihe von Chimären hergestellt, in denen unterschiedlich lange Bereiche der HCV 5’ NTR 3’ terminal mit der IRES des Poliovirus fusioniert wurden. Mit diesem Ansatz konnten wir zeigen, dass die ersten 120 Nukleotide der HCV 5’ NTR als Minimaldomäne für Replikation ausreichen. Weiterhin ergab sich eine klare Korrelation zwischen der Länge der HCV 5’ NTR und der Replikationseffizienz. Mit steigender Länge der 5’ NTR nahm auch die Replikationseffizienz zu, die dann maximal war, wenn das vollständige 5’ Element mit der Poliovirus-IRES fusioniert wurde. Die hier gefundene Kopplung von Translation und Replikation in der HCV 5’ NTR könnte auf einen Mechanismus zur Regulation beider Funktionen hindeuten. Es konnte allerdings noch nicht geklärt werden, welche Bereiche innerhalb der Grenzen des IRES-Elements genau für die RNA-Replikation benötigt werden. Untersuchungen im Bereich der 3’ NTR ergaben, dass die variable Region für die Replikation entbehrlich, die X-Sequenz jedoch essentiell ist. Der polyU/UC-Bereich musste eine Länge von mindestens 11-30 Uridinen besitzen, wobei maximale Replikation ab einer Länge von 30-50 Uridinen beobachtet wurde. Die Addition von heterologen Sequenzen an das 3’ Ende der HCV-RNA führte zu einer starken Reduktion der Replikation. In den hier durchgeführten Untersuchungen zeigte keines der Elemente in der 3’ NTR einen signifikanten Einfluss auf die Translation. Ein weiteres cis aktives RNA-Element wurde im 3’ kodierenden Bereich für das NS5B Protein beschrieben. Wir fanden, dass Veränderungen dieser Struktur durch stille Punktmutationen die Replikation hemmten, welche durch die Insertion einer intakten Version dieses RNA-Elements in die variable Region der 3’ NTR wieder hergestellt werden konnte. Dieser Versuchsansatz erlaubte die genaue Untersuchung der für die Replikation kritischen Strukturelemente. Dadurch konnte gezeigt werden, dass die Struktur und die Primärsequenz der Loopbereiche essentiell sind. Darüber hinaus wurde eine Sequenzkomplementarität zwischen dem Element in der NS5B-kodierenden Region und einem RNA-Bereich in der X-Sequenz der 3’ NTR gefunden, die eine sog. „kissing loop“ Interaktion eingehen kann. Mit Hilfe von gezielten Mutationen konnten wir zeigen, dass diese RNA:RNA Interaktion zumindest transient stattfindet und für die Replikation des HCV essentiell ist.

Methionine-independent initiation of translation in the capsid protein of an insect RNA virus

Protein synthesis is believed to be initiated with the amino acid methionine because the AUG translation initiation codon of mRNAs is recognized by the anticodon of initiator methionine transfer RNA. A group of positive-stranded RNA viruses of insects, however, lacks an AUG translation initiation codon for their capsid protein gene, which is located at the downstream part of the genome. The capsid protein of one of these viruses, Plautia stali intestine virus, is synthesized by internal ribosome entry site-mediated translation. Here we report that methionine is not the initiating amino acid in the translation of the capsid protein in this virus. Its translation is initiated with glutamine encoded by a CAA codon that is the first codon of the capsid-coding region. The nucleotide sequence immediately upstream of the capsid-coding region interacts with a loop segment in the stem–loop structure located 15–43 nt upstream of the 5′ end of the capsid-coding region. The pseudoknot structure formed by this base pair interaction is essential for translation of the capsid protein. This mechanism for translation initiation differs from the conventional one in that the initiation step controlled by the initiator methionine transfer RNA is not necessary.

PseudoBase: structural information on RNA pseudoknots

PseudoBase is a database containing structural, functional and sequence data related to RNA pseudo­knots. It can be reached at http://wwwbio.LeidenUniv.nl/∼Batenburg/PKB.html. For each pseudoknot, thirteen items are stored, for example the relevant sequence, the stem positions of the pseudoknot, the EMBL accession number of the sequence and the support that can be given regarding the reliability of the pseudo­knot. Since the last publication, information on sizes of the stems and the loops in the pseudoknots has been added. Also added are alternative entries that produce surveys of where the pseudoknots are, sorted according to stem size or loop size.

Aminoacylation identity switch of turnip yellow mosaic virus RNA from valine to methionine results in an infectious virus.

The turnip yellow mosaic virus genomic RNA terminates at its 3' end in a tRNA-like structure that is capable of specific valylation. By directed mutation, the aminoacylation specificity has been switched from valine to methionine, a novel specificity for viral tRNA-like structures. The switch to methionine specificity, assayed in vitro under physiological buffer conditions with wheat germ methionyl-tRNA synthetase, required mutation of the anticodon loop and the acceptor stem pseudoknot. The resultant methionylatable genomes are infectious and stable in plants, but genomes that lack strong methionine acceptance (as previously shown with regard to valine acceptance) replicate poorly. The results indicate that amplification of turnip yellow mosaic virus RNA requires aminoacylation, but that neither the natural (valine) specificity nor interaction specifically with valyl-tRNA synthetase is crucial.

Alternate Conformations Regulate Ribosomal Recoding in a Positive-sense RNA Virus

Positive-sense RNA viruses are important animal, plant, insect and bacteria pathogens and constitute the largest group of RNA viruses. Due to the relatively small size of their genomes, these viruses have evolved a variety of non-canonical translation mechanisms to optimize coding capacity expanding their proteome diversity. One such strategy is codon redefinition or recoding. First described in viruses, recoding is a programmed translation event in which codon alterations are context dependent. Recoding takes place in a subset of messenger RNA (mRNAs) with some products reflecting new, and some reflecting standard, meanings. The ratio between the two is both critical and highly regulated. While a variety of recoding mechanisms have been documented, (ribosome shunting, stop-carry on, termination-reinitiation, and translational bypassing), the two most extensively employed by RNA viruses are Programmed Ribosomal Frameshifting (PRF) and Programmed Ribosomal Readthrough (PRT). While both PRT and PRF subvert normal decoding for expression of C-terminal extension products, the former involves an alteration of reading frame, and the latter requires decoding of a non-sense codon. Both processes occur at a low but defined frequency, and both require Recoding Stimulatory Elements (RSE) for regulation and optimum functionality. These stimulatory signals can be embedded in the RNA in the form of sequence or secondary structure, or trans-acting factors outside the mRNA such as proteins or micro RNAs (miRNA). Despite 40+ years of study, the precise mechanisms by which viral RSE mediate ribosome recoding for the synthesis of their proteins, or how the ratio of these products is maintained, is poorly defined. This study reveals that in addition to a long distance RNA:RNA interaction, three alternate conformations and a phylogenetically conserved pseudoknot regulate PRT in the carmovirus Turnip crinkle virus (TCV).

Lineage replacement accompanying duplication and rapid fixation of an RNA element in the nsP3 gene in a species of alphavirus

A sequence of thirty-six nucleotides in the nsP3 gene of Ross River virus (RRV), coding for the amino acid sequence HADTVSLDSTVS, was duplicated some time between 1969 and 1979 coinciding with the appearance of a new lineage of this virus and with a major outbreak of Epidemic Polyarthritis among residents of the Pacific Islands. This lineage of RRV continues to circulate throughout Australia and both earlier lineages, which lacked the duplicated element, now are extinct. Multiple copies of several other elements also were observed in this region of the nsP3 gene in all lineages of RRV. Multiple copies of one of these, coding for the amino acid sequence P*P*PR, were detected in the C-terminal region of the nsP3 protein of all alphaviruses except those of African origin. The fixation of duplications and insertions in 3' region of nsP3 genes from all lineages of alphaviruses, suggests they provide some fitness advantage

Physical and functional interaction of the archaeal single-stranded DNA-binding protein SSB with RNA polymerase

Archaeal transcription utilizes a complex multisubunit RNA polymerase and the basal transcription factors TBP and TF(II)B, closely resembling its eukaryal counterpart. We have uncovered a tight physical and functional interaction between RNA polymerase and the single-stranded DNA-binding protein SSB in Sulfolobus solfataricus. SSB stimulates transcription from promoters in vitro under TBP-limiting conditions and supports transcription in the absence of TBP. SSB also rescues transcription from repression by reconstituted chromatin. We demonstrate the potential for promoter melting by SSB, suggesting a plausible basis for the stimulation of transcription. This stimulation requires both the single-stranded DNA-binding domain and the acidic C-terminal tail of the SSB. The tail forms a stable interaction with RNA polymerase. These data reveal an unexpected role for single-stranded DNA-binding proteins in transcription in archaea.

Identification of a cryptic Prokaryotic promoter within the cDNA encoding the 5′ End of Dengue Virus RNA Genome

Infectious cDNA clones of RNA viruses are important research tools, but flavivirus cDNA clones have proven difficult to assemble and propagate in bacteria. This has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. Here we identify and characterize an efficient cryptic bacterial promoter in the cDNA encoding the dengue virus (DENV) 5′ UTR. Following cryptic transcription in E. coli, protein expression initiated at a conserved in-frame AUG that is downstream from the authentic DENV initiation codon, yielding a DENV polyprotein fragment that was truncated at the N-terminus. A more complete understanding of constitutive viral protein expression in E. coli might help explain the cloning and propagation difficulties generally observed with flavivirus cDNA.