Canonical Termination Research Articles

Internal Transcription Terminators Control Stoichiometry of ABC Transporters in Cellulolytic Clostridia.

ABSTRACTThe extracellular substrate-binding proteins (SBPs) of ATP-binding cassette (ABC) importers tend to be expressed in excess relative to their cognate translocators, but how the stoichiometry of ABC transporters is controlled remains unclear. Here, we elucidated a mechanism contributing to differential gene expression in operons encoding ABC importers by employing cellulolytic Clostridia species, specifically Ruminiclostridium cellulolyticum. We found that there were usually stem-loop structures downstream of SBP genes, which could prematurely terminate the transcription of ABC importers and were putative internal intrinsic terminators, resulting in high transcript levels of upstream SBP genes and low transcript levels of downstream cognate translocator genes. This was determined by their termination efficiencies. Internal terminators had a lower U content in their 3′ U-rich tracts and longer GC-rich stems, which distinguishes them from canonical terminators and potentially endows them with special termination efficiencies. The pairing of U-rich tracts and the formation of unpaired regions in these internal terminators contributed to their folding energies, affecting the stability of their upstream SBP transcripts. Our findings revealed a strategy of internal transcriptional terminators controlling in vivo stoichiometry of their flanking transcripts.IMPORTANCE Operons encoding protein complexes or metabolic pathways usually require fine-tuned gene expression ratios to create and maintain the appropriate stoichiometry for biological functions. In this study, a strategy for controlling differential expression of genes in an operon was proposed by utilizing ABC importers from Ruminiclostridium cellulolyticum. We found that a stem-loop structure is introduced into the intergenic regions of operons encoding ABC importers as the putative internal terminator, which results in the premature termination of transcription. Consequently, the stoichiometric ratio of genes flanking terminators is precisely determined by their termination efficiencies and folding energies at the transcriptional level. Thus, it can be utilized as a promising synthetic biology tool to control the differential expression of genes in an operon.

Case Report: Successful Management of a 29-Day-Old Infant With Severe Hyperlipidemia From a Novel Homozygous Variant of GPIHBP1 Gene

BackgroundSevere hyperlipidemia is characterized by markedly elevated blood triglyceride levels and severe early-onset cardiovascular diseases, pancreatitis, pancreatic necrosis or persistent multiple organ failure if left untreated. It is a rare autosomal recessive metabolic disorder originated from the variants of lipoprotein lipase gene, and previous studies have demonstrated that most cases with severe hyperlipidemia are closely related to the variants of some key genes for lipolysis, such as LPL, APOC2, APOA5, LMF1, and GPIHBP1. Meanwhile, other unidentified causes also exist and are equally worthy of attention.MethodsThe 29-day-old infant was diagnosed with severe hyperlipidemia, registering a plasma triglyceride level as high as 25.46 mmol/L. Whole exome sequencing was conducted to explore the possible pathogenic gene variants for this patient.ResultsThe infant was put on a low-fat diet combined with pharmacological therapy, which was successful in restraining the level of serum triglyceride and total cholesterol to a low to medium range during the follow-ups. The patient was found to be a rare novel homozygous duplication variant-c.45_48dupGCGG (Pro17Alafs*22) in GPIHBP1 gene-leading to a frameshift which failed to form the canonical termination codon TGA. The mutant messenger RNA should presumably produce a peptide consisting of 16 amino acids at the N-terminus, with 21 novel amino acids on the heels of the wild-type protein.ConclusionsOur study expands on the spectrum of GPIHBP1 variants and contributes to a more comprehensive understanding of the genetic diagnosis, genetic counseling, and multimodality therapy of families with severe hyperlipidemia. Our experience gained in this study is also contributory to a deeper insight into severe hyperlipidemia and highlights the importance of molecular genetic tests.

The complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) with implication of phylogenomics and population genetics.

In this study, the complete mitogenome of Lysmata vittata (Crustacea: Decapoda: Hippolytidae) has been determined. The genome sequence was 22003 base pairs (bp) and it included thirteen protein-coding genes (PCGs), twenty-two transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and three putative control regions (CRs). The nucleotide composition of AT was 71.50%, with a slightly negative AT skewness (-0.04). Usually the standard start codon of the PCGs was ATN, while cox1, nad4L and cox3 began with TTG, TTG and GTG. The canonical termination codon was TAA, while nad5 and nad4 ended with incomplete stop codon T, and cox1 ended with TAG. The mitochondrial gene arrangement of eight species of the Hippolytidae were compared with the order of genes of Decapoda ancestors, finding that the gene arrangement order of the Lebbeus groenlandicus had not changed, but the gene arrangement order of other species changed to varying degrees. The positions of the two tRNAs genes (trnA and trnR) of the L. vittata had translocations, which also showed that the Hippolytidae species were relatively unconserved in evolution. Phylogenetic analysis of 50 shrimp showed that L. vittata formed a monophyletic clade with Lysmata/Exhippolysmata species. This study should be helpful to better understand the evolutionary status, and population genetic diversity of L. vittata and related species.

Characterization and Phylogenetic Analysis of the Mitochondrial Genome Sequence of Nisia fuliginosa (Hemiptera: Fulgoroidea: Meenoplidae).

We explored characterization of the mitochondrial genome (mitogenome or mtGenome) and phylogenetic analysis between 32 Fulgoroid species by sequencing and analyzing the mitogenome of Nisia fuliginosa Yang and Hu, 1985 (Hemiptera: Fulgoroidea: Meenoplidae), thereby making it the first determined mitogenome from the family Meenoplidae. The mitogenome was found to be 15,754 bp in length and contained 13 protein-coding genes (PCGs), 22 tRNA genes, two ribosomal RNA genes (rRNAs), and a control region. All PCGs started with typical ATN codons, except for nad1, which used GTG as the start codon. Canonical TAA termination codons were found in 10 PCGs and the remaining three genes (cox2, nad6, and nad1) had incomplete stop codons T. All tRNAs could fold into typical cloverleaf secondary structures, with the exception of trnC, trnV, and trnS1. Additionally, we compared the AT and GC skews of 13 PCGs of 32 Fulgoroidea mitogenomes, on the L-strand, the AT and GC skews were negative and positive, respectively. However, on the H-strand, the AT skew could be positive or negative and the GC skew was always negative. Phylogenetic results showed that the eight families of Fulgoroidea were divided into two large groups. Delphacidae formed a monophyletic group sister to a clade comprising Meenoplidae and other six families (Fulgoridae, Ricaniidae, Flatidae, Issidae, Caliscelidae, and Achilidae). Meenoplidae was located near the clade of Delphacidae, and Fulgoridae was located near the clade of Meenoplidae. Furthermore, Caliscelidae, Issidae, Ricaniidae, and Flatidae are closely related and they collectively formed a sister group to Achilidae.

Repurposing tRNAs for nonsense suppression

Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the TΨC-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 Å resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon.

Termi-Luc: a versatile assay to monitor full-protein release from ribosomes.

Termination of protein biosynthesis is an essential step of gene expression, during which a complete functional protein is released from the ribosome. Premature or inefficient termination results in truncated, nonfunctional, or toxic proteins that may cause disease. Indeed, more than 10% of human genetic diseases are caused by nonsense mutations leading to premature termination. Efficient and sensitive approaches are required to study eukaryotic termination mechanisms and to identify potential therapeutics that modulate termination. Canonical radioactivity-based termination assays are complex, report on a short peptide release, and are incompatible with high-throughput screening. Here we describe a robust and simple in vitro assay to study the kinetics of full-protein release. The assay monitors luminescence upon release of nanoluciferase from a mammalian pretermination complex. The assay can be used to record time-progress curves of protein release in a high-throughput format, making it optimal for studying release kinetics and for high-throughput screening for small molecules that modulate the efficiency of termination.

A high-resolution map of bacteriophage ϕX174 transcription

Bacteriophage ϕX174 is a model virus for studies across the fields of structural biology, genetics, gut microbiomics, and synthetic biology, but did not have a high-resolution transcriptome until this work. In this study we used next-generation sequencing to measure the RNA produced from ϕX174 while infecting its host E. coli C. We broadly confirm the past transcriptome model while revealing several interesting deviations from previous knowledge. Additionally, we measure the strength of canonical ϕX174 promoters and terminators and discover both a putative new promoter that may be activated by heat shock sigma factors, as well as rediscover a controversial Rho-dependent terminator. We also provide evidence for the first antisense transcription observed in the Microviridae, identify two promoters that may be involved in generating this transcriptional activity, and discuss possible reasons why this RNA may be produced.

Targeted Identification of Protein Interactions in Eukaryotic mRNA Translation.

To identify protein-protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP-MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP-tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross-validation approach is employed to identify the most statistically significant protein-protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae's mRNA translation proteins and complexes are identified.

Rescuing stalled mammalian mitoribosomes - what can we learn from bacteria?

In the canonical process of translation, newly completed proteins escape from the ribosome following cleavage of the ester bond that anchors the polypeptide to the P-site tRNA, after which the ribosome can be recycled to initiate a new round of translation. Not all protein synthesis runs to completion as various factors can impede the progression of ribosomes. Rescuing of stalled ribosomes in mammalian mitochondria, however, does not share the same mechanisms that many bacteria use. The classic method for rescuing bacterial ribosomes is trans-translation. The key components of this system are absent from mammalian mitochondria; however, four members of a translation termination factor family are present, with some evidence of homology to members of a bacterial back-up rescue system. To date, there is no definitive demonstration of any other member of this family functioning in mitoribosome rescue. Here, we provide an overview of the processes and key players of canonical translation termination in both bacteria and mammalian mitochondria, followed by a perspective of the bacterial systems used to rescue stalled ribosomes. We highlight any similarities or differences with the mitochondrial translation release factors, and suggest potential roles for these proteins in ribosome rescue in mammalian mitochondria.

Translation termination on mRNAs lacking a stop codon

Translation of mRNAs often terminates at the end of the coding sequence when a stop codon is encountered. However, in nonstop mRNAs that lack a stop codon, the ribosome stalls at the 3′ end of the mRNA and needs to be rescued. In bacteria, stalled ribosomes are rescued by the transfer‐messenger (tmRNA)‐SmpB, the alternative rescue factor A (ArfA)‐RF2 or ArfB proteins (1–3). Although crystal structures of ribosomes complexed with tmRNA‐SmpB and ArfB were solved, the mechanism and molecular interactions involved in the ArfA‐RF2 pathway remained unclear. Recently, an induced‐fit mechanism has been reported for this pathway (4). Here we present a cryoEM structure of the Escherichia coli 70S ribosome on a nonstop mRNA in the presence of ArfA and methylated RF2 (5). Molecular interactions revealed by the structure show how ArfA recognizes a truncated mRNA and recruits the canonical termination factor RF2 to rescue the stalled ribosome (Figure 1). The positively charged C terminus of ArfA occupies the empty mRNA entry channel on the 30S subunit. Consistent with our kinetic and biochemical data, specific molecular interactions show how ArfA recruits RF2, not RF1, to the stalled ribosome. The N‐terminal domain of ArfA interacts with the switch loop of RF2, directing the universally conserved methylated Gly‐Gly‐Glnm (GGQm) motif of RF2 into the peptidyl‐transferase center (PTC) of the ribosome. Furthermore, the conformation of the methylated GGQm motif was determined, which shows that the post‐translational modification stabilizes the side chain of glutamine and thus helps orient the carbonyl oxygen of the glutamine towards the catalytic water molecule. Taken together, we determined the molecular mechanism and interactions important for the ArfA/RF2‐mediated ribosome rescue on nonstop mRNAs and provide insights into the function of the methyl group on the GGQ motif in translation termination on the ribosome.Support or Funding InformationThis study was supported from the National Institute of General Medical Sciences of the NIH (R01‐GM120552). The computational part of the study was supported by the National Institute of General Medical Sciences (P41‐GM104601 to J.C.P. and E.T.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Conformation of methylated GGQ in the Peptidyl Transferase Center during Translation Termination

The universally conserved Gly-Gly-Gln (GGQ) tripeptide in release factors or release factor-like surveillance proteins is required to catalyze the release of nascent peptide in the ribosome. The glutamine of the GGQ is methylated post-translationally at the N5 position in vivo; this covalent modification is essential for optimal cell growth and efficient translation termination. However, the precise conformation of the methylated-GGQ tripeptide in the ribosome remains unknown. Using cryoEM and X-ray crystallography, we report the conformation of methylated-GGQ in the peptidyl transferase center of the ribosome during canonical translational termination and co-translation quality control. It has been suggested that the GGQ motif arose independently through convergent evolution among otherwise unrelated proteins that catalyze peptide release. The requirement for this tripeptide in the highly conserved peptidyl transferase center suggests that the conformation reported here is likely shared during termination of protein synthesis in all domains of life.

High-resolution transcription maps reveal the widespread impact of roadblock termination in yeast.

Transcription termination delimits transcription units but also plays important roles in limiting pervasive transcription. We have previously shown that transcription termination occurs when elongating RNA polymerase II (RNAPII) collides with the DNA-bound general transcription factor Reb1. We demonstrate here that many different DNA-binding proteins can induce termination by a similar roadblock (RB) mechanism. We generated high-resolution transcription maps by the direct detection of RNAPII upon nuclear depletion of two essential RB factors or when the canonical termination pathways for coding and non-coding RNAs are defective. We show that RB termination occurs genomewide and functions independently of (and redundantly with) the main transcription termination pathways. We provide evidence that transcriptional readthrough at canonical terminators is a significant source of pervasive transcription, which is controlled to a large extent by RB termination. Finally, we demonstrate the occurrence of RB termination around centromeres and tRNA genes, which we suggest shields these regions from RNAPII to preserve their functional integrity.

Structure of the 40S-ABCE1 post-splitting complex in ribosome recycling and translation initiation.

The essential ATP-binding cassette protein ABCE1 splits 80S ribosomes into 60S and 40S subunits after canonical termination or quality-control-based mRNA surveillance processes. However, the underlying splitting mechanism remains enigmatic. Here, we present a cryo-EM structure of the yeast 40S-ABCE1 post-splitting complex at 3.9-Å resolution. Compared to the pre-splitting state, we observe repositioning of ABCE1's iron-sulfur cluster domain, which rotates 150° into a binding pocket on the 40S subunit. This repositioning explains a newly observed anti-association activity of ABCE1. Notably, the movement implies a collision with A-site factors, thus explaining the splitting mechanism. Disruption of key interactions in the post-splitting complex impairs cellular homeostasis. Additionally, the structure of a native post-splitting complex reveals ABCE1 to be part of the 43S initiation complex, suggesting a coordination of termination, recycling, and initiation.

Structural basis for ArfA-RF2-mediated translation termination on mRNAs lacking stop codons.

In bacteria, ribosomes stalled on truncated mRNAs that lack a stop codon are rescued by the transfer-messenger RNA (tmRNA), alternative rescue factor A (ArfA) or ArfB systems. Although tmRNA-ribosome and ArfB-ribosome structures have been determined, how ArfA recognizes the presence of truncated mRNAs and recruits the canonical termination release factor RF2 to rescue the stalled ribosomes is unclear. Here we present a cryo-electron microscopy reconstruction of the Escherichia coli 70S ribosome stalled on a truncated mRNA in the presence of ArfA and RF2. The structure shows that the C terminus of ArfA binds within the mRNA entry channel on the small ribosomal subunit, and explains how ArfA distinguishes between ribosomes that bear truncated or full-length mRNAs. The N terminus of ArfA establishes several interactions with the decoding domain of RF2, and this finding illustrates how ArfA recruits RF2 to the stalled ribosome. Furthermore, ArfA is shown to stabilize a unique conformation of the switch loop of RF2, which mimics the canonical translation termination state by directing the catalytically important GGQ motif within domain 3 of RF2 towards the peptidyl-transferase centre of the ribosome. Thus, our structure reveals not only how ArfA recruits RF2 to the ribosome but also how it promotes an active conformation of RF2 to enable translation termination in the absence of a stop codon.

Distinct types of translation termination generate substrates for ribosome-associated quality control.

Cotranslational degradation of polypeptide nascent chains plays a critical role in quality control of protein synthesis and the rescue of stalled ribosomes. In eukaryotes, ribosome stalling triggers release of 60S subunits with attached nascent polypeptides, which undergo ubiquitination by the E3 ligase Ltn1 and proteasomal degradation facilitated by the ATPase Cdc48. However, the identity of factors acting upstream in this process is less clear. Here, we examined how the canonical release factors Sup45–Sup35 (eRF1–eRF3) and their paralogs Dom34-Hbs1 affect the total population of ubiquitinated nascent chains associated with yeast ribosomes. We found that the availability of the functional release factor complex Sup45–Sup35 strongly influences the amount of ubiquitinated polypeptides associated with 60S ribosomal subunits, while Dom34-Hbs1 generate 60S-associated peptidyl-tRNAs that constitute a relatively minor fraction of Ltn1 substrates. These results uncover two separate pathways that target nascent polypeptides for Ltn1-Cdc48-mediated degradation and suggest that in addition to canonical termination on stop codons, eukaryotic release factors contribute to cotranslational protein quality control.

The complete mitochondrial genome of the Neophocaena asiaeorientails sunamrei (Phocaenidae: Neophocaena)

The complete sequence of the mitochondrial DNA (mtDNA) of the East Asian finless porpoise (Neophocaena asiaeorientails sunamrei) has been completed and annotated in this study. The circular genome is 16 385 bp in length, consisting of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 non-coding areas. Ten protein-coding genes use ATG as the initiation codon, except that ND2 started with ATT, and ND3 and ND5 started with ATA. Canonical TAA and TAG termination codons are found in seven protein-coding genes, the remaining four (ND4, ND2, ND3, and COIII) have incomplete termination codons (T or TA), in addition, the COI ends with AGG, and the Cyt b ends with AGA. On the neighbor-joining phylogenetic tree, these results show that the relationship of Phocoenidae is closer with Delphinidae.

Peptide release promoted by methylated RF2 and ArfA in nonstop translation is achieved by an induced-fit mechanism.

Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2m), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on kcat by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the kcat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.

Base Flipping by MTERF1 Can Accommodate Multiple Conformations and Occurs in a Stepwise Fashion

Human mitochondrial transcription termination occurs within the leu-tRNA gene and is mediated by the DNA binding protein MTERF1. The crystal structure of MTERF1 bound to the canonical termination sequence reveals a rare base flipping event that involves the eversion of three nucleotides. These nucleotides are stabilized by stacking interactions with three MTERF1 residues, which are essential not only for base flipping but also for termination activity. To further understand the mechanism of base flipping, we examined each of the individual stacking interactions in structural, energetic and functional detail. Individual substitutions of Arg162, Tyr288 and Phe243 have revealed unequal contributions to overall termination activity. Furthermore, our work identifies an important role for Phe322 in the base flipping mechanism and we demonstrate how Phe322 and Phe243 are important for coupling base flipping between the heavy and light strand DNA chains. We propose a stepwise model for the base flipping process that recapitulates our observations. Finally, we show that MTERF1 has the ability to accommodate alternate active conformations. The adaptability of base flipping has implications for MTERF1 function and for the putative function of MTERF1 at alternative binding sites in human mitochondria.

The complete mitochondrial genome of the orb-weaver spider Cyclosa argenteoalba Boes. et Str. (Araneae: Araneidae)

The complete mitochondrial genome of an orb-weaver spider Cyclosa argenteoalba is determined in this article. The genome encodes the same 37 genes with a length of 14,575 bp as all other metazoan mitogenomes. The A + T content of the majority strand is 73.1%. Among the protein-coding genes, one gene (COI) begins with TTA, three (COII, COIII and ND6) start with ATT, three (ATP6, ND3 and ND4) begin with ATA and other six genes use ATT as initiation codon. All the protein-coding genes end with the canonical termination codons (TAA or TAG), except for ND3 and ND4L, which use an incomplete stop codon (T). Thirteen transfer RNAs genes lack the potential to form the cloverleaf-shaped secondary structure. The A + T-rich region is 1052 bp in length with an A + T content of 68.9%. In the phylogenetic analyses, C. argenteoalba was nested within Araneidae and the species from the superfamily Araneoidea form a momophyletic group.

Spermine Specifically Inhibits the RRF (ribosome recycling factor) Reaction: In its presence, RRF still Stimulates Protein Synthesis

Although spermine (Spm) is not present in E. coli, 100 µM Spm inhibited the disassembly of the model PoTC 100%. On the other hand, in vitro incorporation of amino acids programmed by synthetic mRNA similar to naturally occurring mRNA with canonical initiation and termination signals was stimulated 100% by RRF even in the presence of 100 µM of Spm. These observations indicate that RRF functions to stimulate protein synthesis at steps in addition to the recycling step; our current hypothesis is that RRF stimulates the translocation step. To test this, conventional assay of the translocation as well as the single ribosome translocation step are examined. The inhibitory action of Spm on the RRF reaction is at steps of the release of mRNA and tRNA but not on the binding or release of RRF to and from the ribosome. The inhibitory effect of Spm was partially reversed by IF3. In E. coli mutant unable to metabolize spermidine in vivo, 25% increase of RRF amount was observed. Furthermore, addition of 4 mM spermidine to the culture media of this mutant raised in vivo concentration of RRF about 100%.