Protein structure and the sequential structure of mRNA: α-Helix and β-sheet signals at the nucleotide level

A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed. We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting protein. The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain. A complete search for GenBank nucleotide sequences coding for structural entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment. By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets. These signals do not originate from the clustering of rare codons, but from the similarity of codons coding for very abundant amino acid residues at the N- and C-termini of helices and sheets. No correlation between the positioning of rare codons and the location of structural units was found. The mRNA signals were also compared with conserved nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome. © 1996 Wiley-Liss, Inc.     

Deciphering the rules by which dynamics of mRNA secondary structure affect translation efficiency in Saccharomyces cerevisiae

Messenger RNA (mRNA) secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of mRNA secondary structure is assumed to be reduced in highly translated mRNAs. However, previous studies in vitro reported a positive correlation between mRNA folding strength and protein abundance. The counterintuitive finding suggests that mRNA secondary structure affects translation efficiency in an undetermined manner. Here, we analyzed the folding behavior of mRNA during translation and its effect on translation efficiency. We simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and mRNA secondary structures. We showed that mRNA secondary structure shortens ribosomal distance through the dynamics of folding strength. Notably, when adjacent ribosomes are close, mRNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of mRNA secondary structure and codon usage in highly translated mRNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of mRNA secondary structure coupling with codon usage affect translation efficiency.     

The secondary structure and poly(A) content of globin messenger RNA as a pure RNA and in polyribosome-derived ribonucleoprotein complexes.

The conformation in solution of duck and rabbit globin mRNA, and of the duck mRNA in the mRNA - protein particle, has been investigated by optical methods and also by the use of the dye ethidium bromide which becomes highly fluorescent when intercalated into the double-stranded regions of a nucleic acid. On the basis of the properties of this dye and on the ability of homopolyribonucleotides to form double-stranded structures we have, in addition, developed a simple and sensitive assay for the detection and quantitisation of sequences rich in a particular residue that may be present in an RNA chain. In solution, 45 to 60% of the nucleotides of duck globin nRNA were found to be in bihelical regions. A similar degree of secondary structure was found in rabbit globin mRNA (this paper), as well as in calf lens mRNA and mRNAs from ewe mammary gland (other results). All samples of globin mRNA examined in this work containeda sequence of poly(A), which has poly(U) binding properties similar to that of synthetic poly(a): no specific interaction between the poly(A) sequence and the rest of the molecules can be detected. The fraction of adenosine residues within these poly(A) segments represents 4% in rabbit mRNA and 8 to 9% in duck mRNA. An additional adenosine-rich segment interspersed with guanosine and possibly other residues, was also detected in one duck mRNA sample. The RNA in the duck mRNA - protein particle is also highly structured. The melting profile in the range of 20 to 65 degrees C is quite similar to that of free mRNA and the ability of ethidium bromide to intercalate is reduced to the extent of 70%. Yet the dichroic spectra of free and bound mRNA are significantly distinct. These data suggest that free and protein-bound mRNA May have a very similar degree of secondary structure but with distinct detailed conformation in bihelical regions (change in base tilting for example). Direct evidence has been obtained that proteins stick to the poly(A) segment in the particle since the fraction of adenosine residues detectable by our poly(u) titration procedure is reduced to 50% of that observed in the free mRNA.     

Mimicking the action of folding chaperones in molecular dynamics simulations: Application to the refinement of homology-based protein structures

A novel method for the refinement of misfolded protein structures is proposed in which the properties of the solvent environment are oscillated in order to mimic some aspects of the role of molecular chaperones play in protein folding in vivo. Specifically, the hydrophobicity of the solvent is cycled by repetitively altering the partial charges on solvent molecules (water) during a molecular dynamics simulation. During periods when the hydrophobicity of the solvent is increased, intramolecular hydrogen bonding and secondary structure formation are promoted. During periods of increased solvent polarity, poorly packed regions of secondary structures are destabilized, promoting structural rearrangement. By cycling between these two extremes, the aim is to minimize the formation of long-lived intermediates. The approach has been applied to the refinement of structural models of three proteins generated by using the ROSETTA procedure for ab initio structure prediction. A significant improvement in the deviation of the model structures from the corresponding experimental structures was observed. Although preliminary, the results indicate computationally mimicking some functions of molecular chaperones in molecular dynamics simulations can promote the correct formation of secondary structure and thus be of general use in protein folding simulations and in the refinement of structural models of small- to medium-size proteins.     

RNA secondary structure and compensatory evolution

The classic concept of epistatic fitness interactions between genes has been extended to study interactions within gene regions, especially between nucleotides that are important in maintaining pre-mRNA/mRNA secondary structures. It is shown that the majority of linkage disequilibria found within the Drosophila Adh gene are likely to be caused by epistatic selection operating on RNA secondary structures. A recently proposed method of RNA secondary structure prediction based on DNA sequence comparisons is reviewed and applied to several types of RNAs, including tRNA, rRNA, and mRNA. The patterns of covariation in these RNAs are analyzed based on Kimura's compensatory evolution model. The results suggest that this model describes the substitution process in the pairing regions (helices) of RNA secondary structures well when the helices are evolutionarily conserved and thermodynamically stable, but fails in some other cases. Epistatic selection maintaining pre-mRNA/mRNA secondary structures is compared to weak selective forces that determine features such as base composition and synonymous codon usage. The relationships among these forces and their relative strengths are addressed. Finally, our mutagenesis experiments using the Drosophila Adh locus are reviewed. These experiments analyze long-range compensatory interactions between the 5' and 3' ends of Adh mRNA, the different constraints on secondary structures in introns and exons, and the possible role of secondary structures in RNA splicing.     

Nonuniform size distribution of nascent peptides. The effect of messenger RNA structure upon the rate of translation.

The size distribution of bacteriophage MS2 coat protein nascent chains purified from MS2-infected Escherichia coli has been determined. Accumulations of nascent chains of discrete sizes were observed, providing evidence that the rate of chain elongation during coat protein biosynthesis is not uniform. A correlation of the size of nascent peptides which accumulate during MS2 coat protein biosynthesis and the position on the MS2 coat protein mRNA of the ribosome carrying those lengths of nascent peptides may be made. This correlation leads to the hypothesis that regions of mRNA secondary structure impede the movement of ribosomes during chain elongation and thus serve as the origin of the accumulation of nascent chains of discrete sizes.     

Reinvestigating the codon and amino acid usage of S. cerevisiae genome: a new insight from protein secondary structure analysis

Biased usage of synonymous codons has been elucidated under the perspective of cellular tRNA abundance for quite a long time now. Taking advantage of publicly available gene expression data for Saccharomyces cerevisiae, a systematic analysis of the codon and amino acid usages in two different coding regions corresponding to the regular (helix and strand) as well as the irregular (coil) protein secondary structures, have been performed. Our analyses suggest that apart from tRNA abundance, mRNA folding stability is another major evolutionary force in shaping the codon and amino acid usage differences between the highly and lowly expressed genes in S. cerevisiae genome and surprisingly it depends on the coding regions corresponding to the secondary structures of the encoded proteins. This is obviously a new paradigm in understanding the codon usage in S. cerevisiae. Differential amino acid usage between highly and lowly expressed genes in the regions coding for the irregular protein secondary structure in S. cerevisiae is expounded by the stability of the mRNA folded structure. Irrespective of the protein secondary structural type, the highly expressed genes always tend to encode cheaper amino acids in order to reduce the overall biosynthetic cost of production of the corresponding protein. This study supports the hypothesis that the tRNA abundance is a consequence of and not a reason for the biased usage of amino acid between highly and lowly expressed genes.     

Correlation between mRNA structure of the coding region and translational pauses.

Discontinuous translational elongation of polypeptides is observed during spider dragline silk fibroin synthesis (1,2). The repeating segment of one of the two subunit proteins constituting spider major ampullate (dragline) silk of Nephila clavipes, Spidroin 2, consists of alternate alanine-rich and proline-rich regions (3). It was found that the calculated free energy of the secondary structure of Spidroin 2 mRNA per nucleotide for the alanine-rich region is about the same as that for the successive proline-rich region. The small stability changes of local mRNA secondary structures along the mRNA chain suggest that the translational pauses observed for dragline silk fibroin synthesis may not be correlated with Spidroin 2 mRNA structure, in contrast to Spidroin 1 mRNA structure which may explain the translational pauses (4).     

The context analysis of polynucleotide sequences. II. Inverted repeats and complementary palindromes in RNA-polymerase genes

A new approach to the reconstruction of the RNA secondary structure is suggested on the basis of the method of contextual analysis of polynucleotide sequences. The coding gene regions of beta-, beta'-, sigma-subunits of E. coli RNA polymerase and of phage T7 RNA polymerase were analysed. The clusters of non-random inverted repeats were found in all these genes. The mRNA coded by them can be folded into compact secondary structures. The latter are formed by quite long helices with a few cases of mispairing.     

Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution

Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the ‘long-term evolution experiment’. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins.     

A model for messenger RNA sequences maximizing secondary structure due to code degeneracy.

The general occurrence of stable self-complementary mRNAs in the cells of higher organisms is assumed. As an example the amino acid sequence of human α-globin was “retranslated” into a hypothetical polynucleotide sequence, corrected as much as possible by mutant globins and completed by the chain termination variant “Constant Spring”. Under the idea of optimizing secondary structures due to code degeneracy models for α-globin mRNA with base paired regions were constructed on the basis of thermodynamic data. They were chosen by hand and by a Fortran program. Preservation of mRNA conformations is discussed as a possible function of code degeneracy during evolution.     

Mechanical property of a TIM-barrel protein

The mechanical response of a TIM-barrel protein to an applied pressure has been studied. We generated structures under an applied pressure by assuming the volume change to be a linear function of normal mode variables. By Delaunay tessellation, the space occupied by protein atoms is divided uniquely into tetrahedra, whose four vertices correspond to atomic positions. Based on the atoms that define them, the resulting Delaunay tetrahedra are classified as belonging to various secondary structures in the protein. The compressibility of various regions identified with respect to secondary structural elements in this protein is obtained from volume changes of respective regions in two structures with and without an applied pressure. We found that the β barrel region located at the core of the protein is quite soft. The interior of the β barrel, occupied by side chains of β strands, is the softest. The helix, strand, and loop segments themselves are extremely rigid, while the regions existing between these secondary structural elements are soft. These results suggest that the regions between secondary structural elements play an important role in protein dynamics. Another aspect of tetrahedra, referred to as bond distance, is introduced to account for rigidities of the tetrahedra. Bond distance is a measure of separation of the atoms of a tetrahedron in terms of number of bonds along the polypeptide chain or side chains. Tetrahedra with longer bond distances are found to be softer on average. From this behavior, we derive a simple empirical equation, which well describes the compressibilities of various regions. © 1997 Wiley-Liss Inc.     

In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs

A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo.The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules.     

mRNA环结构对蛋白质折叠速率的影响

前期的相关研究发现mRNA二级结构中存在对蛋白质折叠速率的重要影响因素.而mRNA二级结构中普遍存在着各种复杂的环结构,这些环结构是否对蛋白质折叠速率也有重要的影响呢?不同的环结构对蛋白质折叠速率的影响是否相同呢?基于此想法,建立了一个包含mRNA内部环、发夹环、膨胀环和多分支环等环结构信息和相应蛋白质折叠速率的数据库.对于数据库中的每一个蛋白质,计算了mRNA二级结构中各种环结构碱基含量、配对碱基含量及单链碱基含量等参量,分析了各参量与相应蛋白质折叠速率的相关性.结果显示,各种环结构碱基含量与蛋白质折叠速率均呈极显著或显著正相关.说明mRNA环结构对蛋白质折叠速率有重要的影响.进一步,把蛋白质按照不同折叠类型或不同二级结构类型分组后,对每一组蛋白质重复上述的分析工作.结果表明,对不同类蛋白质,mRNA的各种环结构对其相应蛋白质折叠速率的影响存在着显著差异.上述研究将为进一步开展有关mRNA和蛋白质折叠速率的研究奠定理论基础.     

Gene expression in the polycistronic operons of Escherichia coli heat-labile toxin and cholera toxin: a new model of translational control

A new model is proposed based on the suggestion that stable local secondary structures of mRNA interfere with ribosome movement on mRNA and consequently reduce the translation rate. This model accounts for a different level of translation for each cistron in the polycistronic mRNA of Escherichia coli heat-labile toxin (LT) and cholera toxin. We also conclude that the mRNA secondary structures have been conserved during the evolution of the toxin genes for its functional importance.     

A supersecondary structure library and search algorithm for modeling loops in protein structures

We present a fragment-search based method for predicting loop conformations in protein models. A hierarchical and multidimensional database has been set up that currently classifies 105,950 loop fragments and loop flanking secondary structures. Besides the length of the loops and types of bracing secondary structures the database is organized along four internal coordinates, a distance and three types of angles characterizing the geometry of stem regions. Candidate fragments are selected from this library by matching the length, the types of bracing secondary structures of the query and satisfying the geometrical restraints of the stems and subsequently inserted in the query protein framework where their fit is assessed by the root mean square deviation (r.m.s.d.) of stem regions and by the number of rigid body clashes with the environment. In the final step remaining candidate loops are ranked by a Z-score that combines information on sequence similarity and fit of predicted and observed phi/psi main chain dihedral angle propensities. Confidence Z-score cut-offs were determined for each loop length that identify those predicted fragments that outperform a competitive ab initio method. A web server implements the method, regularly updates the fragment library and performs prediction. Predicted segments are returned, or optionally, these can be completed with side chain reconstruction and subsequently annealed in the environment of the query protein by conjugate gradient minimization. The prediction method was tested on artificially prepared search datasets where all trivial sequence similarities on the SCOP superfamily level were removed. Under these conditions it is possible to predict loops of length 4, 8 and 12 with coverage of 98, 78 and 28% with at least of 0.22, 1.38 and 2.47 A of r.m.s.d. accuracy, respectively. In a head-to-head comparison on loops extracted from freshly deposited new protein folds the current method outperformed in a approximately 5:1 ratio an earlier developed database search method.     

Ribosome-mediated translational pause and protein domain organization.

Because regions on the messenger ribonucleic acid differ in the rate at which they are translated by the ribosome and because proteins can fold cotranslationally on the ribosome, a question arises as to whether the kinetics of translation influence the folding events in the growing nascent polypeptide chain. Translationally slow regions were identified on mRNAs for a set of 37 multidomain proteins from Escherichia coli with known three-dimensional structures. The frequencies of individual codons in mRNAs of highly expressed genes from E. coli were taken as a measure of codon translation speed. Analysis of codon usage in slow regions showed a consistency with the experimentally determined translation rates of codons; abundant codons that are translated with faster speeds compared with their synonymous codons were found to be avoided; rare codons that are translated at an unexpectedly higher rate were also found to be avoided in slow regions. The statistical significance of the occurrence of such slow regions on mRNA spans corresponding to the oligopeptide domain termini and linking regions on the encoded proteins was assessed. The amino acid type and the solvent accessibility of the residues coded by such slow regions were also examined. The results indicated that protein domain boundaries that mark higher-order structural organization are largely coded by translationally slow regions on the RNA and are composed of such amino acids that are stickier to the ribosome channel through which the synthesized polypeptide chain emerges into the cytoplasm. The translationally slow nucleotide regions on mRNA possess the potential to form hairpin secondary structures and such structures could further slow the movement of ribosome. The results point to an intriguing correlation between protein synthesis machinery and in vivo protein folding. Examination of available mutagenic data indicated that the effects of some of the reported mutations were consistent with our hypothesis.