The preferential codon usages in variable and constant regions of immunoglobulin genes are quite distinct from each other.

The pattern of codon utilization in the variable and constant regions of immunoglobulin genes are compared. It is shown that, in these regions, codon utilizations are quite distinct from one another: For most degenerate codons, there is a selective bias that prefers C and/or G ending codons to U and/or A ending codons in the constant region compared with the bias in the variable region. This would strongly suggest that, in immunoglobulin genes, the bias in code word usage is determined by other factors than those concerning with the translational mechanism such as tRNA availability and codon-anticodon interaction. A possibility is also suggested that this differance of code word usage between them is due to the existence of secondary structure in the constant region but not in the variable region.     

黄瓜花叶病毒卫星RNA人工突变体的构建及其侵染性测定

从1个369nt的黄瓜花叶病毒(Cucumbermosaicvirus,CMV)卫星RNA的cDNA出发,采用DNA改组技术构建人工突变体,经过体外转录,将其与不携带卫星RNA的黄瓜花叶病毒株进行假重组,鉴定突变体的生物活性,结果显示所获得的4个卫星RNA的点突变子MS1、MS5、MS6和MS11中,只有MS11仍然具有复制能力;而其他3个点突变子,尽管均只有1个位点的替换,却不能在辅助病毒作用下复制。序列比较分析发现MS11的突变位点位于卫星RNA变异区内,发生的突变与自发突变一致,其他3个突变子的突变位点发生在卫星RNA的高度保守区。而且通过侵染性试验证实突变子MS11与野生型Yi没有明显的差异。由此可推测卫星RNA序列中的高度保守区与卫星RNA的生物活性密切相关,个别碱基的突变会导致RNA二级结构的改变,进而引起其复制能力或稳定性的完全丧失。     

On the accessibility and selection of the initiator site of mRNA in protein synthesis.

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the "accessibility" of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.     

On the accessibility and selection of the initiator site of mRNA in protein synthesis

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the “accessibility” of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.     

Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA. An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.     

Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells

A protein of the C group, most likely C3 (Mr approximately 42,000, pI approximately 6, corresponding to IEF 48m,n of the HeLa protein catalogue (Celis, J. E., Bravo, R., Arenstorf, H. P., and LeStourgeon, W. M. (1986) FEBS Lett. 194, 101-109)), a minor hnRNP protein was purified to near homogeneity under nondenaturing conditions from 40 S heterogeneous nuclear ribonucleoprotein particles. Type C protein stoichiometrically disrupts the residual secondary structure of natural and synthetic RNAs, e.g. HeLa hnRNA, coliphage MS2 RNA, and poly(rU)-spermine, and decreases the Tm of duplex structures, e.g. poly[r(A + U)], by about 30 degrees C. Binding of the protein to polynucleotides is not highly cooperative and has a stoichiometry of one protein per about 10 nucleotides. Binding experiments with a variety of synthetic and natural poly- and oligonucleotides, including those containing consensus splice site sequences, indicate that the protein has a high affinity for G-rich and U-rich regions, G-rich regions being preferred. Base analogs I and T have affinities for the protein that are similar to G and U. There is little or no affinity for A- and C-rich regions. The presence of A residues in a G- or U-rich sequence does not interfere with binding while C-rich regions decrease or prevent the binding of the protein. The nucleotide specificity of type C protein, e.g. selective binding to an oligonucleotide from the 3' end of an intron, is discussed in relationship to the abundance of G and U and the relative scarcity of C residues in the processing signals in pre-mRNA.     

Characteristics of Nucleotide Substitution in the Hepatitis C Virus Genome: Constraints on Sequence Change in Coding Regions at Both Ends of the Genome

Comparison of complete genome sequences for different variants of hepatitis C virus (HCV) reveals several different constraints on sequence change. Synonymous changes are suppressed in coding regions at both 5′ and 3′ ends of the genome. No evidence was found for the existence of alternative reading frames or for a lower mutation frequency in these regions. Instead, suppression may be due to constraints imposed by RNA secondary structures identified within the core and NS5b genes. Nonsynonymous substitutions are less frequent than synonymous ones except in the hypervariable region of E2 and, to a lesser extent, in E1, NS2, and NS5b. Transitions are more frequent than transversions, particularly at the third position of codons where the bias is 16:1. In addition, nucleotide substitutions may not occur symmetrically since there is a bias toward G or C at the third position of codons, while T ↔ C transitions were twice as frequent as A ↔ G transitions. These different biases do not affect the phylogenetic analysis of HCV variants but need to be taken into account in interpreting sequence change in longitudinal studies. Received: 9 September 1996 / Accepted: 20 April 1997     

Codon usage and secondary structure of MS2 phage RNA.

MS2 is an RNA bacteriophage (3569 bases). The secondary structure of the RNA has been determined, and is known to play an important role in regulating translation. Paired regions of the genome have a higher G+C content than unpaired regions. It has been suggested that this reflects selection for high G+C content to encourage pairing, but a re-analysis of the data together with computer simulation suggest that it is an automatic consequence in any RNA sequence of the way it folds up to minimise its free energy. It has also been suggested that the three registers in which pairing can occur in a coding region are used differentially to optimise the use of the redundancy of the genetic code, but re-analysis of the data shows only weak statistical support for this hypothesis.     

Structure of SL4 RNA from the HIV-1 packaging signal.

The NMR-based structure is described for an RNA model of stem-loop 4 (SL4) from the HIV-1 major packaging domain. The GAGA tetraloop adopts a conformation similar to the classic GNRA form, although there are differences in the details. The type II tandem G.U pairs have a combination of wobble and bifurcated hydrogen bonds where the uracil 2-carbonyl oxygen is hydrogen-bonded to both G,H1 and G,H2. There is the likelihood of a Na(+) ion coordinated to the four carbonyl oxygens in the major groove for these G.U pairs and perhaps to the N7 lone pairs of the G bases as well. A continuous stack of five bases extends over nearly the whole length of the stem to the base of the loop in the RNA 16mer: C15/U14/G13/G5/C6. There is no evidence for a terminal G.A pair; instead, G1 appears quite unrestrained, and A16 stacks on both C15 and G2. Residues G2 through G5 exhibit broadened resonances, especially G3 and U4, suggesting enhanced mobility for the 5'-side of the stem. The structure shows G2/G3/U4 stacking along the same strand, nearly isolated from interaction with the other bases. This is probably an important factor in the signal broadening and apparent mobility of these residues and the low stability of the 16mer hairpin against thermal denaturation.     

The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems

The G x U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G x C or A x U base pairs. The G x U wobble base pair also has unique chemical, structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites containing G x U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.     

Studies on the bacteriophage MS2. XXII. Conformation of MS2 RNA in acid medium

MS2 RNA, which sediments at 27S in a neutral buffer, can be converted to a compact 57S conformation at pH 3.8. Requirements for this conversion, besides protonation, are small concentrations of Mg⁺⁺ ions and a low ionic strength. On the other hand, after heating in the presence of EDTA and at low ionic strength, the RNA can be unfolded to an 11.7S form at pH 6.8 and to 10.5S at pH 3.8. The compact 57S form has lost at least 50% of its secondary structure, as determined by its hypochromicity. It corresponds to a monomer species, as will be shown in a following paper (XXIV). Comparative studies with the homopolymers poly A and poly C and with the heteropolymers poly A,U, poly A,C, and poly A,G indicate that the interactions involved in the acid RNA conformation are not simply explainable by the known interactions of the A–A⁺, C–C⁺, and/or A–C⁺ type.     

5S RNA structure and interaction with transcription factor A. 1. Ribonuclease probe of the structure of 5S RNA from Xenopus laevis oocytes

The structure of Xenopus laevis oocyte (Xlo) 5S ribosomal RNA has been probed with single-strand-specific ribonucleases T1, T2, and A with double-strand-specific ribonuclease V1 from cobra venom. The digestion of 5'- or 3'-labeled renatured 5S RNA samples followed by gel purification of the digested samples allowed the determination of primary cleavage sites. Results of these ribonuclease digestions provide support for the generalized 5S RNA secondary structural model derived from comparative sequence analysis. However, three putative single-stranded regions of the molecule exhibited unexpected V1 cuts, found at C36, U73, U76, and U102. These V1 cuts reflect additional secondary structural features of the RNA including A.G base pairs and support the extended base pairing in the stem containing helices IV and V which was proposed by Stahl et al. [Stahl, D. A., Luehrsen, K. R., Woese, C. R., & Pace, N. R. (1981) Nucleic Acids Res. 9, 6129-6137]. A conserved structure for helix V having a common unpaired uracil residue at Xlo position 84 is proposed for all eukaryotic 5S RNAs. Our results are compared with nuclease probes of other 5S RNAs.     

Motif prediction in ribosomal RNAs Lessons and prospects for automated motif prediction in homologous RNA molecules

The traditional way to infer RNA secondary structure involves an iterative process of alignment and evaluation of covariation statistics between all positions possibly involved in basepairing. Watson-Crick basepairs typically show covariations that score well when examples of two or more possible basepairs occur. This is not necessarily the case for non-Watson-Crick basepairing geometries. For example, for sheared (trans Hoogsteen/Sugar edge) pairs, one base is highly conserved (always A or mostly A with some C or U), while the other can vary (G or A and sometimes C and U as well). RNA motifs consist of ordered, stacked arrays of non-Watson-Crick basepairs that in the secondary structure representation form hairpin or internal loops, multi-stem junctions, and even pseudoknots. Although RNA motifs occur recurrently and contribute in a modular fashion to RNA architecture, it is usually not apparent which bases interact and whether it is by edge-to-edge H-bonding or solely by stacking interactions. Using a modular sequence-analysis approach, recurrent motifs related to the sarcin-ricin loop of 23S RNA and to loop E from 5S RNA were predicted in universally conserved regions of the large ribosomal RNAs (16S- and 23S-like) before the publication of high-resolution, atomic-level structures of representative examples of 16S and 23S rRNA molecules in their native contexts. This provides the opportunity to evaluate the predictive power of motif-level sequence analysis, with the goal of automating the process for predicting RNA motifs in genomic sequences. The process of inferring structure from sequence by constructing accurate alignments is a circular one. The crucial link that allows a productive iteration of motif modeling and realignment is the comparison of the sequence variations for each putative pair with the corresponding isostericity matrix to determine which basepairs are consistent both with the sequence and the geometrical data.     

The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers

The activity of the SR protein family of splicing factors in constitutive or alternative splicing requires direct interactions with the pre-mRNA substrate. Thus it is important to define the high affinity targets of the various SR species and to evaluate their ability to discriminate between defined RNA targets. We have analyzed the binding specificity of the 30-kDa SR protein 9G8, which contains a zinc knuckle in addition to the RNA binding domain (RBD). Using a SELEX approach, we demonstrate that 9G8 selects RNA sequences formed by GAC triplets, whereas a mutated zinc knuckle variant selects different RNA sequences, centered around a (A/U)C(A/U)(A/U)C motif, indicating that the zinc knuckle is involved in the RNA recognition specificity of 9G8. In contrast, SC35 selects sequences composed of pyrimidine or purine-rich motifs. Analyses of RNA-protein interactions with purified recombinant 30-kDa SR proteins or in nuclear extracts, by means of UV crosslinking and immunoprecipitation, demonstrate that 9G8, SC35, and ASF/SF2 recognize their specific RNA targets with high specificity. Interestingly, the RNA sequences selected by the mutated zinc knuckle 9G8 variant are efficiently recognized by SRp20, in agreement with the fact that the RBD of 9G8 and SRp20 are similar. Finally, we demonstrate the ability of 9G8 and of its zinc knuckle variant, or SRp20, to act as efficient splicing transactivators through their specific RNA targets. Our results provide the first evidence for cooperation between an RBD and a zinc knuckle in defining the specificity of an RNA binding domain.