Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.     

Role of ribosome recycling factor (RRF) in translational coupling

RNA phage GA coat and lysis protein expression are translationally coupled through an overlapping termination and initiation codon UAAUG. Essential for this coupling are the proximity of the termination codon of the upstream coat gene to the initiation codon of the lysis gene (either a <3 nucleotide separation or physical closeness through a possible hairpin structure) but not the Shine-Dalgarno sequence. This suggests that the ribosomes completing the coat gene translation are exclusively responsible for translation of the lysis gene. Inactivation of ribosome recycling factor (RRF), which normally releases ribosomes at the termination codon, did not influence the expression of the reporter gene fused to the lysis gene. This suggests the possibility that RRF may not release ribosomes from the junction UAAUG. However, RRF is essential for correct ribosomal recognition of the AUG codon as the initiation site for the lysis gene.     

枯草杆菌核糖体蛋白质突变对碱性蛋白酶基因表达的影响

用枯草杆菌体外转录-翻译偶联系统检测13种19株枯草杆菌核糖体蛋白质突变对碱性蛋白酶基因表达的影响,发现10种13株核糖体蛋白质突变能影响碱性蛋白酶基因的表达。其中依赖链霉素突变核糖体几乎不能翻译碱性蛋白酶mRNA。依赖链霉素突变在翻译层次抑制碱性蛋白酶基因的表达,但对中性蛋白酶基因的表达没有影响。在碱性蛋白酶mRNA翻译起始区有一个复合二级结构,用体外突变方法破坏其中一个,翻译效率提高8.2倍。依赖链霉素突变和抗链霉素突变核糖体的高级结构不同,与碱性蛋白酶mRNA 5'端片段的亲合力也有差异。由于碱性蛋白酶mRNA翻译起始区的复合二级结构和低起始强度以及依赖链霉素突变核糖体高级结构的改变,使依赖链霉素突变核糖体不能翻译碱性蛋白酶mRNA。     

The study of the functional role of enzymatic modifications in bacterial ribosomes by the system biology approach

In this work, we report the methodology of studies of the role of bacterial ribosome modifications for regulation of gene expression. A modification of some ribosomal components can affect translation of certain mRNAs. Changes of cellular protein composition caused by deletions of genes responsible for ribosome modifications were detected by proteomic analysis. Using reporter constructs we determined the particular stage of gene expression responsible for variations of protein concentrations. After identification of the mRNA, whose translation was influenced by ribosome modifications, we determined the mRNA regions in the wild-type strain and the strain with unmodified ribosomes responsible for the changes observed. The methodology developed can be applied to studying other translational control mechanisms.     

Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed

Trigger factor and DnaK protect nascent protein chains from misfolding and aggregation in the E. coli cytosol, but how these chaperones affect the mechanism of de novo protein folding is not yet understood. Upon expression under chaperone-depleted conditions, multidomain proteins such as bacterial beta-galactosidase (beta-gal) and eukaryotic luciferase fold by a rapid but inefficient default pathway, tightly coupled to translation. Trigger factor and DnaK improve the folding yield of these proteins but markedly delay the folding process both in vivo and in vitro. This effect requires the dynamic recruitment of additional trigger factor molecules to translating ribosomes. While beta-galactosidase uses this chaperone mechanism effectively, luciferase folding in E. coli remains inefficient. The efficient cotranslational domain folding of luciferase observed in the eukaryotic system is not compatible with the bacterial chaperone system. These findings suggest important differences in the coupling of translation and folding between bacterial and eukaryotic cells.     

Site-specific mutagenesis of cDNA clones expressing a poliovirus proteinase

The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio-specific cleavages occur at glutamine-glycine (Q-G) pairs that are recognized by the viral-encoded proteinase 3C (formerly called P3-7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN-III-C3-7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino- and carboxy-terminal Q-G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN-III-C3-7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site-specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy-terminal Q-G cleavage site. The mutant recombinant plasmid (designated pIN-III-C3-mu 10) directs the synthesis of a bacterial-polio precursor polypeptide that is like the wild-type construct (pIN-III-C3-7c). However, unlike the wild-type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino-terminal Q-G site but not at the carboxy-terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self-cleavage at its carboxy-terminal Q-G pair.     

Human papillomavirus type 18 E1 protein is translated from polycistronic mRNA by a discontinuous scanning mechanism

Papillomaviruses are small double-stranded DNA viruses that replicate episomally in the nuclei of infected cells. The full-length E1 protein of papillomaviruses is required for the replication of viral DNA. The viral mRNA from which the human papillomavirus type 18 E1 protein is expressed is not known. We demonstrate that in eukaryotic cells, the E1 protein is expressed from polycistronic mRNA containing E6, E7, and E1 open reading frames (ORFs). The translation of adjacent E7 and E1 ORFs is not associated; it is performed by separate populations of ribosomes. The translation of the downstream E1 gene is preceded by ribosome scanning. Scanning happens at least at the 5' end of the polycistronic mRNA and also approximately 100 bp in front of the E1 gene. Long areas in middle of the mRNA are bypassed by ribosomes, possibly by ribosomal "shunting." Inactivation of short minicistrons in the upstream area of the E1 gene did not change the expression level of the E1 gene.     

Translation at higher than an optimal level interferes with coupling at an intercistronic junction

In pairs of adjacent genes co-transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single-stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild-type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine-Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.     

Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase

Feline calicivirus (FCV), a member of the Caliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for trans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.     

The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes

Ribosomes from Gram-negative bacteria such as Escherichia coli exhibit non-specific translation of bacterial mRNAs. That is, they are able to translate mRNAs from a variety of sources in a manner independent of the "strength" of the Shine-Dalgarno region, in contrast to ribosomes from many Gram-positive bacteria, such as Bacillus subtilis, which show specific translation in only being able to translate other Gram-positive mRNA, or mRNAs that have "strong" Shine-Dalgarno regions. There is an evolutionary correlation between the translational specificity and the absence of a protein analogous to E. coli ribosomal protein S1. The specificity observed with B. subtilis ribosomes is a function of their 30 S subunit which lacks S1; translation of Gram-negative mRNA can occur with heterologous ribosomes containing the 30 S subunit of E. coli ribosomes and the 50 S subunit of B. subtilis ribosomes. However, the addition of E. coli S1 alone to B. subtilis ribosome does not overcome their characteristic inability to translate mRNA from Gram-negative organisms. By contrast, the removal of S1 from E. coli ribosomes results in translational behavior similar to that shown by B. subtilis ribosomes in that the S1-depleted E. coli ribosomes can translate mRNA from Gram-positive sources in the absence of added S1, although addition of S1 stimulates further translation of such mRNAs by the E. coli ribosomes.     

Translation of Synthetic and Endogenous Messenger Ribonucleic Acid In Vitro by Ribosomes and Polyribosomes from Clostridium pasteurianum

Ribosomes and polyribosomes from Clostridium pasteurianum were isolated and their activities were compared with those of ribosomes from Escherichia coli in protein synthesis in vitro. C. pasteurianum ribosomes exhibited a high level of activity due to endogenous messenger ribonucleic acid (RNA). For translation of polyuridylic acid [poly(U)], C. pasteurianum ribosomes required a higher concentration of Mg(2+) and a much higher level of poly(U) than did E. coli ribosomes. Phage f2 RNA added to the system with C. pasteurianum ribosomes gave no significant stimulation of protein synthesis in a homologous system or with E. coli initiation factors. The 30S and 50S subunits prepared from C. pasteurianum ribosomes reassociated less readily than subunits from E. coli. The ability of the C. pasteurianum subunits to reassociated was found to be dependent upon the presence of a reducing agent during preparation and during analysis of the reassociation products. In heterologous combinations, E. coli 30S subunits associated readily with C. pasteurianum 50S subunits to form 70S particles, but C. pasteurianum 30S subunits and E. coli 50S subunits did not associate. In poly(U) translation, E. coli 30S subunits were active in combination with 50S subunits from either E. coli or C. pasteurianum, but C. pasteurianum 30S subunits were not active in combination with either type of 50S subunits. Polyribosomes prepared from C. pasteurianum were very active in protein synthesis, and well-defined ribosomal aggregates as large as heptamers could be seen on sucrose gradients. An attempt was made to demonstrate synthesis in vitro of ferredoxin.     

λN gene expression regulated by translation termination in ribosome L24 mutant

Although the initiation and elongation steps of protein synthesis have been extensively char-acterized in Escherichia coli (E. coli), the translation termination is still less well understood. However, recent experiment result might have provided some hints for our deeper understanding of the termination mechanism. (i) Not only does the translation stop codon act as a signal for ter-mination, but also its context influences the translation termination[13]; (ii) the structure similar-ity betwee…     

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression.