Formation of a type I collagen RNA dimer by intermolecular base-pairing of a conserved sequence around the translation initiation site.

A symmetrical sequence around the translation initiation site of several collagen genes is highly conserved. Deletions in this sequence increase translational efficiency of an alpha 2(I) collagen - CAT chimeric gene after DNA transfection of NIH 3T3 fibroblasts (Schmidt, Rossi and de Crombrugghe, submitted). The secondary structure, predicted by the sequence of this segment, was shown to exist in solution in 200 mM NaCl at 37 degrees C. Cell-free translation of the corresponding RNA using a reticulocyte lysate is inhibited 2 to 4-fold by preincubation with a 0.5 M NaCl extract of an NIH 3T3 ribosomal eluate. Cell-free translation of two mutant RNAs, with partial deletions of the conserved sequence, is not inhibited by such preincubation. This inhibition is not due to degradation of the RNA and requires a protein component of the ribosomal eluate, which, however, is not required after the preincubation step. Preincubation of the RNA with the ribosomal eluate from NIH 3T3 fibroblasts causes the reversible formation of an intermolecular dimer in which the conserved symmetrical sequences hybridize to each other. This results in an increase in the degree of secondary structure of the conserved segment around the translation initiation site. We speculate that translational efficiency could be modulated by influencing the equilibrium between monomer and dimer.     

Information and sequence extraction around the 5'-end and translation initiation site of human genes

FIE (5'-end Information Extraction) is a web-based program designed primarily to extract the sequence of the regions around the 5'-end and around the translation initiation sites for a particular gene, based on information provided by LocusLink.     

A conserved nucleotide sequence, coding for a segment of the C-propeptide, is found at the same location in different collagen genes.

The nucleotide sequence of a segment of the chick alpha 1 type III collagen gene which codes for the C-propeptide was determined and compared with the corresponding sequence in the alpha 1 type I and alpha 2 type I collagen genes. As in the alpha 2 type I gene the coding information for the C-propeptide of the type III collagen gene is subdivided in four exons. Similarly, the amino proximal exon contains sequences for both the carboxy terminal end of the alpha-helical segment of collagen and for the beginning of the C-propeptide in both genes. Therefore, this organization of exons must have been established before these two collagen genes arose by duplication of a common ancestor. In several subsegments the deduced amino acid sequence for the C-propeptide of type III collagen shows a strong homology with the corresponding amino acid sequence in alpha 1 and alpha 2 type I. For one of these homologous amino acid sequences, however, the nucleotide sequence is much better conserved than for the others. It is possible that a mechanism of gene conversion has maintained the homogeneity of this nucleotide sequence among the interstitial collagen genes. Alternatively, the conserved nucleotide sequence may represent a regulatory signal which could function either in the DNA or in the RNA.     

鸡基因组差异表达基因翻译起始密码子上下游序列的比较研究

翻译起始调控是基因表达调控的一个关键步骤之一。本文以鸡为研究材料,比较研究了鸡基因组高表达基因和低表达基因翻译起始密码子上下游的碱基序列差异,旨在寻找影响鸡基因表达水平的特异性调控位点。全部3 020个单剪接基因完整的mRNA序列及有详细注释的5＇UTRs序列从Ensembl下载。编写计算机程序,读取每个基因mRNA起始密码子上下游各位点的碱基。研究发现,起始密码子上游-3、-2位点可能是鸡基因组基因表达起始密码子正确识别的关键位点。起始密码子上下游的碱基组成分析发现,高表达基因和低表达基因起始密码子的上游均倾向使用（G＋C）,高表达基因的使用偏倚尤为强烈。序列差异比较发现,高表达基因在-9、-6、-3、＋4位点显著偏向G,在-1、-2、-4、-5位点显著偏向C。低表达基因起始密码子上游使用A、U的频率显著高于低表达基因。在-19位点强烈偏向A,在＋1、＋11、＋14位点强烈偏向U。     

The gene encoding translation initiation factor 3 is highly conserved in gram-negative bacteria

A 1.1-kb Hp alpha I fragment of the Escherichia coli chromosome containing the gene for translation initiation factor 3 was employed as a probe in heterologous hybridization to chromosomal DNA from a variety of other procaryotes. Positive hybridization was observed to DNA derived from all gram-negative bacteria tested. In contrast, no hybridization to DNA from gram-positive bacteria was detected. In addition, homologous sequences were found in Euglena gracilis chloroplast DNA, while this was not the case with Saccharomyces cerevisiae mitochondrial DNA. These results are discussed in light of existing data on the components and mechanism of translation initiation in the various organisms and organelles employed in this study.     

iTIS-PseTNC: A sequence-based predictor for identifying translation initiation site in human genes using pseudo trinucleotide composition

Translation is a key process for gene expression. Timely identification of the translation initiation site (TIS) is very important for conducting in-depth genome analysis. With the avalanche of genome sequences generated in the postgenomic age, it is highly desirable to develop automated methods for rapidly and effectively identifying TIS. Although some computational methods were proposed in this regard, none of them considered the global or long-range sequence-order effects of DNA, and hence their prediction quality was limited. To count this kind of effects, a new predictor, called “iTIS-PseTNC,” was developed by incorporating the physicochemical properties into the pseudo trinucleotide composition, quite similar to the PseAAC (pseudo amino acid composition) approach widely used in computational proteomics. It was observed by the rigorous cross-validation test on the benchmark dataset that the overall success rate achieved by the new predictor in identifying TIS locations was over 97%. As a web server, iTIS-PseTNC is freely accessible at http://lin.uestc.edu.cn/server/iTIS-PseTNC. To maximize the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web server to obtain the desired results without the need to go through detailed mathematical equations, which are presented in this paper just for the integrity of the new prection method.     

A conserved helical element is essential for internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by cap-independent internal ribosome binding to the 5' noncoding region (NCR). To identify the sequences and structural elements within the 5' NCR of HCV RNA that contribute to the initiation of translation, a series of point mutations was introduced within this sequence. Since the pyrimidine-rich tract is considered a characteristic feature of picornavirus internal ribosome entry site (IRES) elements, our mutational analysis focused on two putative pyrimidine tracts (Py-I and Py-II) within the HCV 5' NCR. Translational efficiency of these mutant RNAs was examined by in vitro translation and after RNA transfection into liver-derived cells. Mutational analysis of Py-I (nucleotides 120 to 130), supported by compensatory mutants, demonstrates that the primary sequence of this motif is not important but that a helical structural element associated with this region is critical for HCV IRES function. Mutations in Py-II (nucleotides 191 to 199) show that this motif is dispensable for IRES function as well. Thus, the pyrimidine-rich tract motif, which is considered as an essential element of the picornavirus IRES elements, does not appear to be a functional component of the HCV IRES. Further, the insertional mutagenesis study suggests a requirement for proper spacing between the initiator AUG and the upstream structures of the HCV IRES element for internal initiation of translation.     

A conserved structural motif at the N terminus of bacterial translation initiation factor IF2

The 18-kDa Domain I from the N-terminal region of translation initiation factor IF2 from Escherichia coli was expressed, purified, and structurally characterized using multidimensional NMR methods. Residues 2-50 were found to form a compact subdomain containing three short beta-strands and three alpha-helices, folded to form a betaalphaalphabetabetaalpha motif with the three helices packed on the same side of a small twisted beta-sheet. The hydrophobic amino acids in the core of the subdomain are conserved in a wide range of species, indicating that a similarly structured motif is present at the N terminus of IF2 in many of the bacteria. External to the compact 50-amino acid subdomain, residues 51-97 are less conserved and do not appear to form a regular structure, whereas residues 98-157 form a helix containing a repetitive sequence of mostly hydrophilic amino acids. Nitrogen-15 relaxation rate measurements provide evidence that the first 50 residues form a well ordered subdomain, whereas other regions of Domain I are significantly more mobile. The compact subdomain at the N terminus of IF2 shows structural homology to the tRNA anticodon stem contact fold domains of the methionyl-tRNA and glutaminyl-tRNA synthetases, and a similar fold is also found in the B5 domain of the phenylalanine-tRNA synthetase. The results of the present work will provide guidance for the design of future experiments directed toward understanding the functional roles of this widely conserved structural domain within IF2.     

A conserved HEAT domain within eIF4G directs assembly of the translation initiation machinery

The X-ray structure of the phylogenetically conserved middle portion of human eukaryotic initiation factor (eIF) 4GII has been determined at 2.4 A resolution, revealing a crescent-shaped domain consisting of ten alpha helices arranged as five HEAT repeats. Together with the ATP-dependent RNA helicase eIF4A, this HEAT domain suffices for 48S ribosomal complex formation with a picornaviral RNA internal ribosome entry site (IRES). Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of this essential component of the translation initiation machinery that, respectively, bind eIF4A and a picornaviral IRES. The structural and biochemical results provide mechanistic insights into both cap-dependent and cap-independent translation initiation.     

The +4G site in Kozak consensus is not related to the efficiency of translation initiation

The optimal context for translation initiation in mammalian species is GCCRCCaugG (where R = purine and "aug" is the initiation codon), with the -3R and +4G being particularly important. The presence of +4G has been interpreted as necessary for efficient translation initiation. Accumulated experimental and bioinformatic evidence has suggested an alternative explanation based on amino acid constraint on the second codon, i.e., amino acid Ala or Gly are needed as the second amino acid in the nascent peptide for the cleavage of the initiator Met, and the consequent overuse of Ala and Gly codons (GCN and GGN) leads to the +4G consensus. I performed a critical test of these alternative hypotheses on +4G based on 34169 human protein-coding genes and published gene expression data. The result shows that the prevalence of +4G is not related to translation initiation. Among the five G-starting codons, only alanine codons (GCN), and glycine codons (GGN) to a much smaller extent, are overrepresented at the second codon, whereas the other three codons are not overrepresented. While highly expressed genes have more +4G than lowly expressed genes, the difference is caused by GCN and GGN codons at the second codon. These results are inconsistent with +4G being needed for efficient translation initiation, but consistent with the proposal of amino acid constraint hypothesis.     

Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon.

Hepatitis C viral (HCV) RNA includes an internal ribosome entry segment (IRES) that extends some 30 nt into the coding region and promotes internal initiation of translation at the authentic initiation codon at nt 342. The 5'-boundary of this IRES was mapped by in vitro translation and transfection assays and was found to lie between nt 42 and 71. Within these IRES boundaries there are, in most HCV strains, three AUG triplets upstream of the authentic initiation site. Although the first, 5'-proximal, of these is absolutely conserved, a mutational analysis showed that it is not a functional initiation codon. In particular, the G residue could be substituted provided compensatory mutations were made to maintain base pairing. The other two upstream AUGs are not absolutely conserved, and mutation of the third (5'-distal) had little effect on IRES activity. When an additional AUG codon was introduced by single-site mutation just upstream of the authentic initiation codon, it was found to be used when most of the IRES had been deleted to generate an RNA translated by the scanning ribosome mechanism, but was not used in the background of the full-length IRES when internal initiation is operative. These results argue that the IRES promotes direct ribosome entry immediately at, or indeed very close to, the authentic initiation codon, and that the upstream AUGs do not serve as ribosome entry sites.     

The initiation codon determines the efficiency but not the site of translation initiation in Chlamydomonas chloroplasts.

To study translation initiation in Chlamydomonas chloroplasts, we mutated the initiation codon AUG to AUU, ACG, ACC, ACU, and UUC in the chloroplast petA gene, which encodes cytochrome f of the cytochrome b6/f complex. Cytochrome f accumulated to detectable levels in all mutant strains except the one with a UUC codon, but only the mutant with an AUU codon grew well at 24 degrees C under conditions that require photosynthesis. Because no cytochrome f was detectable in the UUC mutant and because each mutant that accumulated cytochrome f did so at a different level, we concluded that any residual translation probably initiates at the mutant codon. As a further demonstration that alternative initiation sites are not used in vivo, we introduced in-frame UAA stop codons immediately downstream or upstream or in place of the initiation codon. Stop codons at or downstream of the initiation codon prevented accumulation of cytochrome f, whereas the one immediately upstream of the initiation codon had no effect on the accumulation of cytochrome f. These results suggest that an AUG codon is not required to specify the site of translation initiation in chloroplasts but that the efficiency of translation initiation depends on the identity of the initiation codon.     

Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes

Understanding regulatory mechanisms of protein synthesis in eukaryotes is essential for the accurate annotation of genome sequences. Kozak reported that the nucleotide sequence GCCGCC(A/G)CCAUGG (AUG is the initiation codon) was frequently observed in vertebrate genes and that this 'consensus' sequence enhanced translation initiation. However, later studies using invertebrate, fungal and plant genes reported different 'consensus' sequences. In this study, we conducted extensive comparative analyses of nucleotide sequences around the initiation codon by using genomic data from 47 eukaryote species including animals, fungi, plants and protists. The analyses revealed that preferred nucleotide sequences are quite diverse among different species, but differences between patterns of nucleotide bias roughly reflect the evolutionary relationships of the species. We also found strong biases of A/G at position -3, A/C at position -2 and C at position +5 that were commonly observed in all species examined. Genes with higher expression levels showed stronger signals, suggesting that these nucleotides are responsible for the regulation of translation initiation. The diversity of preferred nucleotide sequences around the initiation codon might be explained by differences in relative contributions from two distinct patterns, GCCGCCAUG and AAAAAAAUG, which implies the presence of multiple molecular mechanisms for controlling translation initiation.