5' untranslated leader sequences of eukaryotic mRNAs encoding heat shock induced proteins.

5' untranslated leaders (5' UTLs) are suggested to play a crucial role in the selective translation of their eukaryotic mRNAs encoding heat shock proteins (HSP) during heat stress conditions. However, the structural features of the HSP mRNAs which cause this effect are mostly unknown. We have compiled the 5' UTLs from about 140 eukaryotic HSP mRNAs including vertebrates, invertebrates, higher and lower plants. A detailed analysis of these sequences according to length, A+T content, context of functional ATGs and presence of upstream non-functional ATGs was made. We observed that all these features were similar to the earlier studies in the literature based on data from HSP as well as non-HSP mRNAs. These observations were reconfirmed by intra-specific comparison of 5' UTLs from HSP and non-HSP genes. Similar to the translation element involved in the selective translation of mRNAs in polioviruses, a search for a short sequence motif complementary to highly conserved 18S rRNA was performed using a HSP mRNA database. The majority of the HSP mRNA sequences (77%) contained one or more small sequence motifs suggesting that they may function as internal ribosome entry sites for selective initiation of translation during heat stress.     

Splicing and transport of eukaryotic mRNAs: a theoretical model

Several years have already elapsed since the first discovery of splicing in eukaryotic mRNAs. In this process sections of the precursor mRNAs are spliced out (these are named introns) and the two remaining excised sites, 5′ and 3′ are ligated to form the mature mRNA chains.Very little is known about the splicing and ligation mechanism or about its location inside the cell. It is known to take place in the nucleus, but it is unknown whether it occurs inside the nuclear matter or on the surface of its membrane. Since nearly all eukaryotic messengers undergo splicing, this is a central question.From the theoretical point of view this is an intriguing problem. A lot of data have recently accumulated which have a bearing on this question. Based on current knowledge, this paper proposes a model in which splicing is carried out on the surface of the nuclear membrane and in concert with transport across it. It is suggested that the enzymes that take part in this process are loosely associated with the membrane pore complex. Evidence and results which are relevant to this question are given and discussed.     

All subgenomic mRNAs of equine arteritis virus contain a common leader sequence.

During the replication of equine arteritis virus (EAV) six subgenomic mRNAs are synthesized. We present evidence that the viral mRNAs form a 3'-coterminal nested set and contain a common leader sequence of 208 nucleotides which is encoded by the 5'-end of the genome. The leader is joined to the bodies of mRNA 5 and 6 at positions defined by the sequence 5' UCAAC 3'. The part of the leader sequence flanking the UCAAC motif is very similar to the 5'-splice site of the Tetrahymena pre-rRNA. A possible internal guide sequence has been identified 43 nucleotides downstream of the leader sequence on the genome. Hybridization analysis shows that all EAV intracellular RNAs contain the leader sequence. These data imply that the viral subgenomic mRNAs are composed of leader and body sequences which are non-contiguous on the genome.     

Minimal catalytic domain of N-acetylglucosaminyltransferase V

UDP-GlcNAc: Manalpha1-6Manbeta-R beta1-6 N-acetylglucosaminyltransferase V (EC 2.4.1.155, GlcNAc-TV) is a Golgi enzyme that substitutes the trimannosyl core in the biosynthetic pathway for complex-type N-linked glycans. GlcNAc-TV activity is regulated by oncogenes frequently activated in cancer cells ( ras, src, and her2/neu ) and by activators of T lymphocytes. Overexpression of GlcNAc-TV in epithelial cells results in morphological transformation, while tumor cell mutants selected for loss of GlcNAc-TV products show diminished malignant potential in mice. In this report, we have expressed and characterized a series of N- and C-terminal deletions of GlcNAc-TV. Portions of GlcNAc-TV sequence were fused at the N-terminal domain to IgG-binding domains of staphylococcal Protein A and expressed in CHOP cells. The secreted fusion proteins were purified by IgG Sepharose affinity chromatography and assayed for enzyme activities. The peptide sequence S(213-740)of GlcNAc-TV was determined to be essential for the catalytic activity, the remaining amino acids comprising a 183 amino acid stem region, a 17 amino acid transmembrane domain and a 12 amino acid cytosolic moiety. Further deletion of 5 amino acids to produce peptide R(218-740)reduced enzyme activity by 20-fold. Similar K(m)and V(max)values for donor and acceptor were observed for peptide S(213-740), the minimal catalytic domain, and peptide Q(39-740), which also included the stem region. Truncation of five amino acids from the C-terminus also resulted in a 20-fold loss of catalytic activity. Secondary structure predictions suggest a high frequency of turns in the stem region, and more contiguous stretches of alpha-helix found in the catalytic domain.     

Minimal catalytic domain of a group I self-splicing intron RNA

The self-splicing intron ribozymes have been regarded as primitive forms of the splicing machinery for eukaryotic pre-mRNAs. The splicing activity of group I self-splicing introns is dependent on an absolutely conserved and exceptionally densely packed core region composed of two helical domains, P3-P7 and P4-P6, that are connected rigidly via base triples. Here we show that a mutant group I intron ribozyme lacking both the P4-P6 domain and the base triples can perform the phosphoester transfer reactions required for splicing at both the 5' and 3' splice sites, demonstrating that the elements required for splicing are concentrated in the stacked helical P3-P7 domain. This finding establishes that the conserved core of the intron consists of two physically and functionally separable components, and we present a model showing the architecture of a prototype of this class of intron and the course of its molecular evolution.     

Crystallization of a soluble,catalytically active form of Escherichia coli leader peptidase

Leader peptidase, a novel serine protease in Escherichia coli, catalyzes the cleavage of the amino-terminal leader sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Here, we report a procedure for the purification and the crystallization of a soluble non-membrane-bound form of leader peptidase (Δ2-75). Crystals were obtained by the sitting-drop vapor diffusion technique using ammonium dihydrogen phosphate as the precipitant. Interestingly, we have found that the presence of the detergent Triton X-100 is required to obtain crystals sufficiently large for X-ray analysis. The crystals belong to the tetragonal space group P4₂2₁2, with unit cell dimensions of a = b = 115 Å and c = 100 Å, and contain 2 molecules per asymmetric unit. This is the first report of the crystallization of a leader (or signal) peptidase. © 1995 Wiley-Liss, Inc.     

真核生物mRNA翻译起始机制研究进展

在真核生物中,mRNA翻译是一个复杂的多步骤过程,包括起始、延伸和终止3个阶段。其中,起始阶段的调控是影响mRNA翻译的关键。目前已经发现,mRNA翻译起始方式有多种,以最早发现的m ⁷G帽依赖性扫描机制最为经典,但当细胞处于逆境,经典起始机制受到抑制时,其他类型的起始机制会将其替代以保证翻译的顺利进行。本文对目前已发现的真核生物mRNA不同翻译起始机制特别是经典起始机制的替代机制进行了综述,旨在为深入认识真核生物基因在翻译水平上的表达调控提供参考。     

Alternative translation start sites and hidden coding potential of eukaryotic mRNAs

It is widely suggested that a eukaryotic mRNA typically contains one translation start site and encodes a single functional protein product. However, according to current points of view on translation initiation mechanisms, eukaryotic ribosomes can recognize several alternative translation start sites and the number of experimentally verified examples of alternative translation is growing rapidly. Also, the frequent occurrence of alternative translation events and their functional significance are supported by the results of computational evaluations. The functional role of alternative translation and its contribution to eukaryotic proteome complexity are discussed.     

Potential open reading frames within 5'-untranslated regions of eukaryotic mRNAs

It is known that 5'-untranslated sequences of eukaryotic mRNA often contain AUG triplets, which may serve as the sites of translation initiation. It is thought that these leader open reading frames can fulfil the regulatory functions and encode functionally active proteins. However, they have been incompletely characterized. The article deals with the context organization of leader reading frames of eukaryotic mRNAs. It has been shown that their characteristics correlate with the position relative to the protein-encoding sequence, which may be associated with the efficiency of initiation of translation.     

Comparative analysis of orthologous eukaryotic mRNAs: potential hidden functional signals

Sequencing of multiple, nearly complete eukaryotic genomes creates opportunities for detecting previously unnoticed, subtle functional signals in non-coding regions. A genome-wide comparative analysis of orthologous sets of mammalian and yeast mRNAs revealed distinct patterns of evolutionary conservation at the boundaries of the untranslated regions (UTRs) and the coding region (CDS). Elevated sequence conservation was detected in ~30 nt regions around the start codon. There seems to be a complementary relationship between sequence conservation in the ~30 nt regions of the 5′-UTR immediately upstream of the start codon and that in the synonymous positions of the 5′-terminal 30 nt of the CDS: in mammalian mRNAs, the 5′-UTR shows a greater conservation than the CDS, whereas the opposite trend holds for yeast mRNAs. Unexpectedly, a ~30 nt region downstream of the stop codon shows a substantially lower level of sequence conservation than the downstream portions of the 3′-UTRs. However, the sequence in this poorly conserved 30 nt portion of the 3′-UTR is non-random in that it has a higher GC content than the rest of the UTR. It is hypothesized that the elevated sequence conservation in the region immediately upstream of the start codon is related to the requirement for initiation factor binding during pre-initiation ribosomal scanning. In contrast, the poorly conserved region downstream of the stop codon could be involved in the post- termination scanning and dissociation of the ribosomes from the mRNA, which requires only the mRNA–ribosome interaction. Additionally, it was found that the choice of the stop codon in mammals, but not in yeasts, and the context in the immediate vicinity of the stop codons in both mammals and yeasts are subject to strong selection. Thus, genome-wide analysis of orthologous gene sets allows detection of previously unrecognized patterns of sequence conservation, which are likely to reflect hidden functional signals, such as ribosomal filters that could regulate translation by modulating the interaction between the mRNA and ribosomes.