Base pairing between cis-acting sequences contributes to template switching during plus-strand DNA synthesis in human hepatitis B virus

Hepadnaviruses utilize two template switches (primer translocation and circularization) during synthesis of plus-strand DNA to generate a relaxed-circular (RC) DNA genome. In duck hepatitis B virus (DHBV) three cis-acting sequences, 3E, M, and 5E, contribute to both template switches through base pairing, 3E with the 3' portion of M and 5E with the 5' portion of M. Human hepatitis B virus (HBV) also contains multiple cis-acting sequences that contribute to the accumulation of RC DNA, but the mechanisms through which these sequences contribute were previously unknown. Three of the HBV cis-acting sequences (h3E, hM, and h5E) occupy positions equivalent to those of the DHBV 3E, M, and 5E. We present evidence that h3E and hM contribute to the synthesis of RC DNA through base pairing during both primer translocation and circularization. Mutations that disrupt predicted base pairing inhibit both template switches while mutations that restore the predicted base pairing restore function. Therefore, the h3E-hM base pairing appears to be a conserved requirement for template switching during plus-strand DNA synthesis of HBV and DHBV. Also, we show that base pairing is not sufficient to explain the mechanism of h3E and hM, as mutating sequences adjacent to the base pairing regions inhibited both template switches. Finally, we did not identify predicted base pairing between h5E and the hM region, indicating a possible difference between HBV and DHBV. The significance of these similarities and differences between HBV and DHBV will be discussed.     

Simulations using random-generated DNA and RNA sequences

Using a very simple computer program written in BASIC, a very large number of random-generated DNA or RNA sequences are obtained. Students individually and as a group use these sequences to perform a wide variety of simulated events including predicting complementary sequences and trans-lational products, evaluating base compositions, determining frequencies of particular triplet codons, and suggesting possible secondary structures.     

Recombination and meiosis.

Although exchanges between sister chromatids are common in mitotic cells, those involving homologous chromosomes are rare. Since recombination between homologues is one of the functions of meiosis, it follows that one aspect of the differentiation of the meiocyte involves the synthesis of proteins or enzymes which facilitate synapsis and exchange. Mutants are known which seem to have constitutive levels of mitotic recombination between homologues, and these may be defective in the mechanism which normally represses mitotic recombination. It has been proposed that one component of the synaptonemal complex (s.c.) is a filamentous pairing protein with DNA binding sites which are base sequence specific. Synapsis occurs because the distribution of these sequences is the same in homologues. When only non-homologous chromosomes are present, as in haploid meiosis, only weak pairing can occur, since the base sequences are largely out of register. Although certain features of recombination at the molecular level are known, none of the models so far proposed suggest an explanation for interference between crossovers. It is suggested that interference may depend on the presence of a limited amount of another DNA binding protein which is specifically located within the s.c. A crossover between naked DNA molecules is initially a weak structure, which must be later converted into a visible and mechanically strong chiasma. It is assumed that this stabilization of a crossover is achieved by the DNA binding protein, which can diffuse freely within the s.c. and bind cooperatively to any recombinant DNA molecules within it. Depletion of the binding protein within the vicinity of a crossover makes it unlikely that the second crossover can be formed nearby.     

Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19.

The interaction of protein SRP19 with the RNA component of human signal recognition particle (SRP) was studied by site-directed mutagenesis of the SRP RNA. The effects of nucleotide changes in the tetranucleotide loop (tetraloop) of helix 6 showed that SRP19 recognizes a tetraloop in a sequence-specific manner. Adenosine 149 at the third position of the tetraloop was essential for binding. In contrast, changes of the base at the second position had no effect. Mutations that disrupt or compensate individual SRP RNA helices were generated to investigate the importance of base pairing and to identify other binding sites. Considerable base pairing was essential in helix 6. Another SRP19-binding site was located in the distal part of helix 8. The primary sequences of the tetraloop-binding protein SR19 and of bacterial ribosomal protein S15 are shown to be similar.     

Protein replication by amino acid pairing

Biological problems concerning the origin of life and the mode of prion replication (Prusiner, 1982) may require protein replication (the synthesis of one protein sequence from another) as part of their solution. It is suggested that complementarity between protein sequences may be determined by amino acid pairing (Root-Bernstein, 1982a). Two mechanisms using the complementarity afforded by amino acid pairing are proposed. Experimental tests of the mechanisms are suggested.     

The topology of hepatitis B virus pregenomic RNA promotes its replication

Previous analysis of hepatitis B virus (HBV) indicated base pairing between two cis-acting sequences, the 5' half of the upper stem of epsilon and phi, contributes to the synthesis of minus-strand DNA. Our goal was to identify other cis-acting sequences on the pregenomic RNA (pgRNA) involved in the synthesis of minus-strand DNA. We found that large portions of the pgRNA could be deleted or substituted without an appreciable decrease in the level of minus-strand DNA synthesized, indicating that most of the pgRNA is dispensable and that a specific size of the pgRNA is not required for this process. Our results indicated that the cis-acting sequences for the synthesis of minus-strand DNA are present near the 5' and 3' ends of the pgRNA. In addition, we found that the first-strand template switch could be directed to a new location when a 72-nucleotide (nt) fragment, which contained the cis-acting sequences present near the 3' end of the pgRNA, was introduced at that location. Within this 72-nt region, we uncovered two new cis-acting sequences, which flank the acceptor site. We show that one of these sequences, named omega and located 3' of the acceptor site, base pairs with phi to contribute to the synthesis of minus-strand DNA. Thus, base pairing between three cis-acting elements (5' half of the upper stem of epsilon, phi, and omega) are necessary for the synthesis of HBV minus-strand DNA. We propose that this topology of pgRNA facilitates first-strand template switch and/or the initiation of synthesis of minus-strand DNA.     

Secondary Structural Elements within the 3′ Untranslated Region of Mouse Hepatitis Virus Strain JHM Genomic RNA

Previously, we characterized two host protein binding elements located within the 3'-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3'-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).     

Rfold: an exact algorithm for computing local base pairing probabilities

MOTIVATION: Base pairing probability matrices have been frequently used for the analyses of structural RNA sequences. Recently, there has been a growing need for computing these probabilities for long DNA sequences by constraining the maximal span of base pairs to a limited value. However, none of the existing programs can exactly compute the base pairing probabilities associated with the energy model of secondary structures under such a constraint. RESULTS: We present an algorithm that exactly computes the base pairing probabilities associated with the energy model under the constraint on the maximal span W of base pairs. The complexity of our algorithm is given by O(NW2) in time and O(N+W2) in memory, where N is the sequence length. We show that our algorithm has a higher sensitivity to the true base pairs as compared to that of RNAplfold. We also present an algorithm that predicts a mutually consistent set of local secondary structures by maximizing the expected accuracy function. The comparison of the local secondary structure predictions with those of RNALfold indicates that our algorithm is more accurate. Our algorithms are implemented in the software named 'Rfold.' AVAILABILITY: The C++ source code of the Rfold software and the test dataset used in this study are available at http://www.ncrna.org/software/Rfold/.     

Empirical comparison of cross-platform normalization methods for gene expression data

Background

The prediction of secondary structure, i.e. the set of canonical base pairs between nucleotides, is a first step in developing an understanding of the function of an RNA sequence. The most accurate computational methods predict conserved structures for a set of homologous RNA sequences. These methods usually suffer from high computational complexity. In this paper, TurboFold, a novel and efficient method for secondary structure prediction for multiple RNA sequences, is presented.

Results

TurboFold takes, as input, a set of homologous RNA sequences and outputs estimates of the base pairing probabilities for each sequence. The base pairing probabilities for a sequence are estimated by combining intrinsic information, derived from the sequence itself via the nearest neighbor thermodynamic model, with extrinsic information, derived from the other sequences in the input set. For a given sequence, the extrinsic information is computed by using pairwise-sequence-alignment-based probabilities for co-incidence with each of the other sequences, along with estimated base pairing probabilities, from the previous iteration, for the other sequences. The extrinsic information is introduced as free energy modifications for base pairing in a partition function computation based on the nearest neighbor thermodynamic model. This process yields updated estimates of base pairing probability. The updated base pairing probabilities in turn are used to recompute extrinsic information, resulting in the overall iterative estimation procedure that defines TurboFold. TurboFold is benchmarked on a number of ncRNA datasets and compared against alternative secondary structure prediction methods. The iterative procedure in TurboFold is shown to improve estimates of base pairing probability with each iteration, though only small gains are obtained beyond three iterations. Secondary structures composed of base pairs with estimated probabilities higher than a significance threshold are shown to be more accurate for TurboFold than for alternative methods that estimate base pairing probabilities. TurboFold-MEA, which uses base pairing probabilities from TurboFold in a maximum expected accuracy algorithm for secondary structure prediction, has accuracy comparable to the best performing secondary structure prediction methods. The computational and memory requirements for TurboFold are modest and, in terms of sequence length and number of sequences, scale much more favorably than joint alignment and folding algorithms.

Conclusions

TurboFold is an iterative probabilistic method for predicting secondary structures for multiple RNA sequences that efficiently and accurately combines the information from the comparative analysis between sequences with the thermodynamic folding model. Unlike most other multi-sequence structure prediction methods, TurboFold does not enforce strict commonality of structures and is therefore useful for predicting structures for homologous sequences that have diverged significantly. TurboFold can be downloaded as part of the RNAstructure package at http://rna.urmc.rochester.edu.     

Conversion of amino-acid sequence in proteins to classical music: search for auditory patterns

We have converted genome-encoded protein sequences into musical notes to reveal auditory patterns without compromising musicality. We derived a reduced range of 13 base notes by pairing similar amino acids and distinguishing them using variations of three-note chords and codon distribution to dictate rhythm. The conversion will help make genomic coding sequences more approachable for the general public, young children, and vision-impaired scientists.     

The cleavage specificity of RNase III.

We determined sites in lambda cII mRNA that are cleaved by RNase III in the presence of lambda OOP antisense RNA, using a series of OOP RNAs with different internal deletions. In OOP RNA-cII mRNA structures containing a potential region of continuous double-stranded RNA bounded by a non-complementary unpaired region, RNase III cleaved the cII mRNA at one or more preferred sites located 10 to 14 bases from the 3'-end of the region of continuous complementarity. Cleavage patterns were almost identical when the presumptive structure was the same continuously double-stranded region followed by a single-stranded bulge and a second short region of base pairing. The sequences of the new cleavage sites show generally good agreement with a consensus sequence derived from thirty-five previously determined cleavage sequences. In contrast, four 'non-sites' at which cleavage is never observed show poor agreement with this consensus sequence. We conclude that RNase III specificity is determined both by the distance from the end of continuous pairing and by nucleotide sequence features within the region of pairing.     

Reading frame switch caused by base-pair formation between the 3'' end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli.

Watson-Crick base pairing is shown to occur between the mRNA and nucleotides near the 3' end of 16S rRNA during the elongation phase of protein synthesis in Escherichia coli. This base-pairing is similar to the mRNA-rRNA interaction formed during initiation of protein synthesis between the Shine and Dalgarno (S-D) nucleotides of ribosome binding sites and their complements in the 1540-1535 region of 16S rRNA. mRNA-rRNA hybrid formation during elongation had been postulated to explain the dependence of an efficient ribosomal frameshift on S-D nucleotides precisely spaced 5' on the mRNA from the frameshift site. Here we show that disruption of the postulated base pairs by single nucleotide substitutions, either in the S-D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing. This result implies that the 3' end of 16S rRNA scans the mRNA very close to the decoding sites during elongation.     

原核表达中优化起始密码下游序列的软件设计与实现

在原核表达中影响外源基因表达效率的因素有很多,这其中翻译起始效率起了非常重要的作用。翻译起始效率又主要受SD序列、SD序列与起始密码子之间的间距、DB(DownstreamBox)序列、mRNA翻译起始区(TIR)的二级结构和稀有密码子等因素的影响。主要针对DB序列和5′端稀有密码子的优化设计了软件。通过计算机对序列进行分析比对后,按照匹配碱基数、匹配位置、密码子使用频率平均值的顺序进行排序,给出一些优化序列,并给出了软件算法。     

RNA Sampler: a new sampling based algorithm for common RNA secondary structure prediction and structural alignment

MOTIVATION: Non-coding RNA genes and RNA structural regulatory motifs play important roles in gene regulation and other cellular functions. They are often characterized by specific secondary structures that are critical to their functions and are often conserved in phylogenetically or functionally related sequences. Predicting common RNA secondary structures in multiple unaligned sequences remains a challenge in bioinformatics research. Methods and RESULTS: We present a new sampling based algorithm to predict common RNA secondary structures in multiple unaligned sequences. Our algorithm finds the common structure between two sequences by probabilistically sampling aligned stems based on stem conservation calculated from intrasequence base pairing probabilities and intersequence base alignment probabilities. It iteratively updates these probabilities based on sampled structures and subsequently recalculates stem conservation using the updated probabilities. The iterative process terminates upon convergence of the sampled structures. We extend the algorithm to multiple sequences by a consistency-based method, which iteratively incorporates and reinforces consistent structure information from pairwise comparisons into consensus structures. The algorithm has no limitation on predicting pseudoknots. In extensive testing on real sequence data, our algorithm outperformed other leading RNA structure prediction methods in both sensitivity and specificity with a reasonably fast speed. It also generated better structural alignments than other programs in sequences of a wide range of identities, which more accurately represent the RNA secondary structure conservations. AVAILABILITY: The algorithm is implemented in a C program, RNA Sampler, which is available at http://ural.wustl.edu/software.html     

Codon and base biases after the initiation codon of the open reading frames in the Escherichia coli genome and their influence on the translation efficiency

Nucleotide sequences around the boundaries of all open reading frames in the Escherichia coli whole genome were analyzed. Characteristic base biases were observed after the initiation codon and before the termination codon. We examined the effect of the base sequence after the initiation codon on the translation efficiency, by introducing mutations after the initiation codon of the E. coli dihydrofolate reductase (DHFR) gene, considering codon and base biases, and using in vitro and in vivo translation systems. In both assay systems, the two most frequent second codons, AAA and AAU, enhanced the translation efficiency compared with the wild type, whereas the effects of lower frequency codons were not significant. Experiments using 16S rRNA variants with mutations in the putative complementary sequence to the region downstream of the initiation codon showed that the translation efficiency of none of the DHFR mutants was affected. These results demonstrate that the statistically most frequent sequences for the second codon enhance translation efficiency, and this effect seems to be independent of base pairing between mRNA and 16S rRNA.     

Functional determinants of the quorum‐sensing non‐coding RNAs and their roles in target regulation

Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1–5, control quorum‐sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem‐loops: the first stem‐loop is crucial for base pairing with a subset of targets. This stem‐loop also protects the Qrr sRNAs from RNase E‐mediated degradation. The second stem‐loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem‐loop plays an accessory role in base pairing and stability. The fourth stem‐loop functions as a rho‐independent terminator. In the quorum‐sensing regulon, Qrr sRNAs‐controlled genes are the most rapid to respond to quorum‐sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing.     

Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box

The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA.