Gene Start Research Articles

Mycobacterium bovis BCG dodecin gene codes a functional protein despite of a start codon mutation

Dodecin is a dodecamer involved in flavin homeostasis, with interesting temperature and osmolarity endurance features in Mycobacterium tuberculosis. A single nucleotide polymorphism in the gene's start codon in BCG, converting ATG to ACG, is predicted to generate a N-terminal shorter isoform, lacking the first 7 amino acids. We previously reported that the shortened recombinant protein has reduced extremophilic features. Here we investigate if within the mycobacterial context dodecin can be produced from both alleles, carrying ATG and ACG start codons. Reporter gene assays using mcherry cloned downstream and in phase to both M.tb and BCG “upstream” regions confirms production of functional proteins. Complementation with both dod alleles similarly enhances M. smegmatis growth after entry into logarithmic phase and exposure to hydrogen peroxide, possibly implicating this protein in oxidative stress response mechanisms. Altogether these data indicate that BCG dodecin is indeed produced, notwithstanding in lower levels compared to M.tb, conferring similar phenotypes, even with the SNP altering the M.tb ATG start codon to the BCG ACG. This protein might be an interesting drug target for the development of new therapeutics against tuberculosis.

OSTIR: open source translation initiation rate prediction.

SummaryTranslation of messenger RNAs into proteins by the ribosome is a fundamental step in gene expression. In bacteria, it is possible to accurately predict the rate of translation initiation from the sequence surrounding a gene’s start codon using thermodynamic models of RNA folding and ribosome binding. These predictions have applications in a range of fields, from systems biology studies that aim to understand and model bacterial physiology to synthetic biology studies that seek to reprogram bacterial cells. For example, metabolic engineers can design ribosome binding site (RBS) sequences to tune the expression of different enzymes in a pathway and thereby optimize the production of a desired chemical compound by cells. OSTIR (Open Source Translation Initiation Rates) is a Python package and command line tool for predicting translation initiation rates in bacteria.

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.

BackgroundDue to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.MethodsHere, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.FindingsWe report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.InterpretationThe results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.FundingThis work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

Non-gradient and genotype-dependent patterns of RSV gene expression.

Respiratory syncytial virus (RSV) is a nonsegmented negative-strand RNA virus (NSV) and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3' promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription extending across the genome, with the highest level of mRNA coming from the most promoter-proximal gene, the first nonstructural (NS1) gene, and mRNA levels from subsequent genes dropping until reaching a minimum at the most promoter-distal gene, the polymerase (L) gene. However, recent reports show non-gradient levels of mRNA, with higher than expected levels from the attachment (G) gene. It is unknown to what extent different transcript stabilities might shape measured mRNA levels. It is also unclear whether patterns of RSV gene expression vary, or show strain- or genotype-dependence. To address this, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and in cotton rats infected with RSV isolates belonging to four genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were non-gradient and genotype-specific, where mRNA levels from the G gene exceeded those from the more promoter-proximal nucleocapsid (N) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. Our results indicate that gene expression from a small but diverse set of RSV genotypes is non-gradient and genotype-dependent. We propose novel models of RSV transcription that can account for non-gradient transcription.

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

Respiratory Syncytial Virus Polymerase Can Initiate Transcription from Position 3 of the Leader Promoter

The mechanisms by which the respiratory syncytial virus (RSV) RNA-dependent RNA polymerase (RdRp) initiates mRNA transcription and RNA replication are poorly understood. A previous study, using an RSV minigenome, suggested that the leader (Le) promoter region at the 3' end of the genome has two initiation sites, one at position +1, opposite the 3' terminal nucleotide of the genome, and a second site at position +3, at a sequence that closely resembles the gene start (GS) signal of the RSV L gene. In this study, we show that the +3 initiation site of the Le is utilized with apparently high frequency in RSV-infected cells and yields small RNA transcripts that are heterogeneous in length but mostly approximately 25 nucleotides (nt) long. Experiments with an in vitro assay in which RSV RNA synthesis was reconstituted using purified RdRp and an RNA oligonucleotide showed that nt 1 to 14 of the Le promoter were sufficient to signal initiation from +3 and that the RdRp could access the +3 initiation site without prior initiation at +1. In a minigenome assay, nucleotide substitutions within the Le to increase its similarity to a GS signal resulted in more-efficient elongation of the RNA initiated from position +3 and a reduction in RNA initiated from the NS1 gene start signal at +45. Taken together, these data suggest a new model for initiation of sequential transcription of the RSV genes, whereby the RdRp initiates the process from a gene start-like sequence at position +3 of the Le.

Complete genome sequence of avian paramyxovirus type 7 (strain Tennessee) and comparison with other paramyxoviruses

The complete genome sequence of avian paramyxovirus serotype 7 (APMV-7) prototype strain dove/Tennessee/4/75 was determined. The genome size is 15,480 nucleotides (nt) long and follows the “rule of six”. The genome contains six non-overlapping genes in the order of 3′-N-P/V/W-M-F-HN-L-5′. The 3′-leader and 5′-trailer sequences of the genome are 55 and 127 nt long, respectively. The first 12 nt of the leader and trailer sequences are complementary to each other. The viral genes are flanked by highly conserved gene-start (GS) and gene-end (GE) transcription signals, and in addition the 3′-leader sequence contains a sequence ( 35AAUUAUUUUUU 45) that is identical to the GE signal present at two of the genes. The genes are separated by intergenic sequences (IGS) ranging between 11 and 70 nt. The phosphoprotein (P) gene contains a conserved RNA editing site (3′-UUUUUCCC-5′) presumed to be involved in the production of V and W proteins. The viral fusion (F) protein has a single basic amino acid at the putative cleavage site ( 101TLPSS R↓F 107); however, the virus did not require exogenous protease for in vitro replication. The virus grew in only a few established cell lines, indicating a restricted host range. Sequence alignment and phylogenetic analysis of the predicted amino acid sequence of APMV-7 proteins with the cognate proteins of the viruses of all five genera of the family Paramyxoviridae showed that APMV-7 is more closely related to APMV-2, -6, -8 than to APMV-1, -3, -4 and -9. The mean death time in embryonated chicken eggs was found to be more than 144 h, indicating APMV-7 to be avirulent for chickens.

Mutational analysis of the gene start sequences of pneumonia virus of mice

The transcriptional start sequence of pneumonia virus of mice is more variable than that of the other pneumoviruses, with five different nine-base gene start (GS) sequences found in the PVM genome. The sequence requirements of the PVM gene start signal, and the efficiency of transcriptional initiation of the different virus genes, was investigated using a reverse genetics approach with a minigenome construct containing two reporter genes. A series of GS mutants were created, where each of the nine bases of the gene start consensus sequence of a reporter gene was changed to every other possible base, and the resulting effect on initiation of transcription was assayed. Nucleotide positions 1, 2 and 7 were found to be most sensitive to mutation whilst positions 4, 5 and 9 were relatively insensitive. The L gene GS sequence was found to have only 20% of the activity of the consensus sequence whilst the published M2 gene start sequence was found to be non-functional. A minigenome construct in which the two reporter genes were separated by the F-M2 gene junction of PVM was used to confirm the presence of two alternative, functional, GS sequences that could both drive the transcription of the PVM M2 gene.

Identification of internal sequences in the 3' leader region of human respiratory syncytial virus that enhance transcription and confer replication processivity.

Previous studies of respiratory syncytial virus have shown that the 44-nucleotide (nt) leader (Le) region is sufficient to initiate RNA replication, producing antigenome RNA, and that the Le and adjoining gene start (GS) signal of the first gene are sufficient to initiate transcription, producing mRNA. A cis-acting element necessary for both transcription and replication was mapped within the first 11 nt at the 3' end of Le. In the present study the remainder of the Le region was mapped to identify sequences important for transcription and replication. A series of minigenomes with mutant Le sequences was generated, and their ability to direct transcription and replication was determined by Northern blot analysis, which examined full-length antigenome and mRNA, and by primer extension analysis, which examined antigenome and mRNA initiation. With regard to transcription, nt 36 to 43, located immediately upstream of the GS signal, were found to be necessary for optimal levels of mRNA synthesis, although the GS signal in conjunction with the 3'-terminal region of Le was sufficient to direct accurate mRNA synthesis initiation. With regard to replication, the first 15 nt of Le were found to be sufficient to direct initiation of antigenome synthesis, but nt 16 to 34 were required in addition for efficient encapsidation and production of full-length antigenome. Analysis of transcripts produced from di- and tricistronic minigenomes indicated that a significant proportion of abortive replicases continue RNA synthesis to the end of the first gene and then continue in a transcription mode along the remainder of the genome.

Functional analysis of the genomic and antigenomic promoters of human respiratory syncytial virus.

The promoters involved in transcription and RNA replication by respiratory syncytial virus (RSV) were examined by using a plasmid-based minireplicon system. The 3' ends of the genome and antigenome, which, respectively, contain the 44-nucleotide (nt) leader (Le) and 155-nt trailer-complement (TrC) regions, should each contain a promoter for RNA replication. The 3' genome end also should have the promoter for transcription. Substitution for the Le with various lengths of TrC demonstrated that the 3'-terminal 36 nt of TrC are sufficient for extensive (but not maximal) replication and that when juxtaposed with a transcription gene-start (GS) signal, this sequence was also able to direct transcription. It was also shown that the region of Le immediately preceding the GS signal of the first gene could be deleted with either no effect or with a slight decrease in transcription initiation. Thus, the TrC is competent to direct transcription even though it does not do so in nature, and the partial sequence identity it shares with the 3' end of the genome likely represents the important elements of a conserved promoter active in both replication and transcription. Increasing the length of the introduced TrC sequence incrementally to 147 nt resulted in a fourfold increase in replication and a nearly complete inhibition of transcription. These two effects were unrelated, implying that transcription and replication are not interconvertible processes mediated by a common polymerase, but rather are independent processes. The increase in replication was specific to the TrC sequence, implying the presence of a nonessential, replication-enhancing cis-acting element. In contrast, the inhibitory effect on transcription was due solely to the altered spacing between the 3' end of the genome and GS signal, which implies that the transcriptase recognizes the first GS signal as a promoter element. Neither the enhancement of replication nor the inhibition of transcription was due to increased base-pairing potential between the 3' and 5' ends. The relative strengths of the Le and TrC promoters for directing RNA synthesis were compared and found to be very similar. Thus, these findings highlighted a high degree of functional similarity between the RSV antigenomic and genomic promoters, but provided a further distinction between promoter requirements for transcription and replication.

Effect of inserting paramyxovirus simian virus 5 gene junctions at the HN/L gene junction: analysis of accumulation of mRNAs transcribed from rescued viable viruses.

Simian parainfluenza virus 5 (SV5) is a prototype of the Paramyxoviridae family of nonsegmented negative-sense RNA viruses. The single-stranded RNA genomes of these viruses contain a series of tandemly linked genes separated by intergenic (IG) sequences flanked by gene-end (GE) and gene-start (GS) sequences. The viral RNA polymerase (vRNAP) complex is thought to enter the genome at its 3' end, and synthesis of mRNAs is thought to occur by a stop-start mechanism in a sequential and polar manner, with transcriptional attenuation occurring primarily at the intergenic regions. As a result, multiple nonoverlapping mRNA species are generated for each single entry of the vRNAP. To investigate the functions of GE, IG, and GS sequences in transcription, we constructed plasmids containing cDNAs of the full-length SV5 genome in which the gene junction sequences (GE, IG, and GS sequences) located between the hemagglutinin-neuraminidase (HN) and the polymerase (L) genes were replaced with the counterpart sequences from other gene junctions. By using reverse genetics, we recovered viable viruses from each cDNA construct, although their growth characteristics varied. Analysis of the HN and L mRNAs by quantitative RNase protection assay indicated that the ratios of HN to L mRNAs varied over a fourfold range. The alteration of the gene junction sequences also permitted examination of the hypothesized requirement for hexamer nucleotide position of the GS sites. The recovery of infectious viruses with transcription initiation sites that occurred at nucleotide positions 1, 2, 3, 5, and 6 of the hexamer suggest that the requirement is nonstringent.

Model for polymerase access to the overlapped L gene of respiratory syncytial virus.

The last two genes of respiratory syncytial virus (RSV), M2 and L, overlap by 68 nucleotides, an arrangement which has counterparts in a number of nonsegmented negative-strand RNA viruses. Thus, the gene-end (GE) signal of M2 lies downstream of the L gene-start (GS) signal, separated by 45 nucleotides. Since RSV transcription ostensibly is sequential and unidirectional from a single promoter within the 3' leader region, it was unclear how the polymerase accesses the L GS signal. Furthermore, it was previously shown that 90% of transcripts which are initiated at the L GS signal are polyadenylated and terminated at the M2 GE signal, yielding a short, truncated L mRNA as the major transcription product of the L gene. Despite these apparent down-regulatory features, we show that the accumulation of full-length L mRNA during RSV infection is only sixfold less than that of its upstream neighbor, M2. We used cDNA-encoded genome analogs in an intracellular transcription assay to investigate the mechanism of transcription of the overlapped genes. Expression of L was found to be dependent on sequential transcription from the 3' end of the genome. Apart from the L GS signal, the only other strict requirement for initiation at L was the M2 GE signal. This implies that the polymerase accesses the L GS signal only following arrival at the M2 GE signal. Thus, polymerase which terminates at the M2 GE signal presumably scans upstream to initiate at the L GS signal. This also would provide a mechanism whereby polymerase which terminates prematurely during transcription of L could recycle from the M2 GE signal to the L GS signal, thereby accounting for the unexpectedly high level of synthesis of full-length L mRNA. The sequence and spacing between the two signals were not critical. Furthermore, the polymerase also was capable of efficiently transcribing from an L GS signal placed downstream of the M2 GE signal, implying that the overlapping arrangement is not obligatory. When copies of the L GS signal were placed concurrently upstream and downstream of the M2 GE signal, both were utilized. This finding indicates that a polymerase situated at a GE signal is capable of scanning for a GS signal in either the upstream or downstream direction and thereafter initiating transcription.

Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA.

The gene start (GS) and gene end (GE) transcription signals of human respiratory syncytial virus (RSV) strain A2 were analyzed in helper-dependent monocistronic and dicistronic minireplicons which were complemented by a standard RSV strain. The GS signal, which is the start site for mRNA synthesis, is highly conserved for the first nine genes: 3'-CCCCGUUUA(U/C) (negative sense). This conserved version of the signal was analyzed by "saturation" mutagenesis, in which all 10 positions, as well as one downstream and one upstream position, were changed one at a time into each of the other three nucleotides. Most of the positions appear to contribute to the signal: positions 1, 3, 6, 7, and, in particular, 9 were the most sensitive, whereas position 5 was relatively insensitive. The effect of nucleotide substitution in the first position of the signal was examined further by cDNA cloning and sequence analysis of the residual mRNA which was produced. For the two mutants examined (1C to U, and 1C to A), the site of initiation was unchanged. However, the mRNAs were dimorphic with regard to the assignment of the 5'-terminal nucleotide: two-thirds contained the predicted mutant substitution, and one-third contained the parental assignment. Intracellular minigenome contained only the mutant assignment, indicating that the heterogeneity was at the level of transcription by the RSV polymerase. This suggests that the templated mutant assignment at position 1 can sometimes be overridden by an innate preference for the parental assignment, a phenomenon which we dubbed quasi-templated initiation. The GS signal of the L gene, encoding the 10th RSV mRNA, contains three differences (3'-CCCUGUUUUA) compared to the conserved version. It was shown to be equal in efficiency to the conserved version. This was unexpected, since the saturation mutagenesis described above indicated that U in place of A at position 9 should be highly inhibitory. Instead, the A at position 10 of the L GS signal was found to be critical for activity, indicating that an essential A residue indeed was present in both versions of the GS signal but that its spacing differed. The GE signal, which directs termination and polyadenylation, has more sequence diversity in nature than does the GS signal. The naturally occurring GE signals of strain A2 were compared by their individual incorporation into a dicistronic minigenome. They were similar in the ability to produce translatable mRNA except in the cases of NS1 and NS2, which were approximately 60% as efficient.

Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus.

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) motifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequential transcription were analyzed with cDNA-encoded RNA analogs (minigenomes) of nonsegmented negative-sense RSV genomic RNA. Two minigenomes were used. The monocistronic RSV-CAT minigenome consists of the chloramphenicol acetyltransferase (CAT) translational open reading frame (ORF) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA. The dicistronic RSV-CAT-LUC minigenome is a derivative of RSV-CAT into which the ORF for luciferase (LUC), bordered by GS and GE motifs, was inserted downstream of the CAT gene with an intergenic region positioned between the two genes. Each minigenome was synthesized in vitro and transfected into RSV-infected cells, where it was replicated and transcribed to yield the predicted polyadenylated subgenomic mRNA(s). The only RSV sequences required for efficient transcription and RNA replication were the 44-nucleotide 3' leader region, the last 40 nucleotides of the 5' trailer region, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable transcription signals which could be attached to foreign sequences to direct their transcription into subgenomic mRNAs. Removal of the GS motif greatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream position. Ablation of the promoter-proximal GS signal was not associated with increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV-CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUC mRNA. The latter result showed that a downstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal. This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry. Removal of both the GS and GE signals of the upstream CAT gene in RSV-CAT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant. Remarkably, whereas both genes were silent when the CAT GS and GE signals were both absent, restoration of the CAT GE signal alone restored a significant level (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequential transcription that was independent of the promoter-proximal GS signal and appeared to involve readthrough from the leader region.

The structurally diverse intergenic regions of respiratory syncytial virus do not modulate sequential transcription by a dicistronic minigenome

The first nine genes of respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus, are separated by intergenic regions which range in size from 1 to 52 nucleotides for strain A2 and lack obvious consensus elements except that each ends in an A (genome sense). Their significance for gene expression was investigated by using RSV-CAT-LUC RNA, a helper-dependent cDNA-encoded dicistronic analog of RSV genomic RNA in which the viral genes were replaced by a negative-sense copy of the translational open reading frame (ORF) encoding chloramphenicol acetyltransferase (CAT) as the upstream, leader-proximal gene and that encoding luciferase (LUC) as the downstream gene. These foreign ORFs were flanked by the RSV gene-start (GS) and gene-end (GE) transcription signals and separated by the naturally occurring G/F intergenic region. The RSV-CAT-LUC minigenome was synthesized in vitro and transfected into RSV-infected cells, and synthesis of the CAT and LUC mRNAs was monitored by enzyme assay and Northern (RNA) blot hybridization. Surprisingly, substitution of each of the other naturally occurring RSV intergenic regions in turn did not significantly alter the absolute or relative amounts of the two mRNAs. Substitution of a nonnatural 10-nucleotide intergenic region, or elimination of the intergenic region altogether, also had little effect on the level of expression of the two genes. Four of the minigenome variants containing naturally occurring intergenic regions were modified further by replacing part of the LUC ORF with a second copy of the CAT ORF, so that each of the two mRNAs would hybridize equally with a CAT-specific probe and their relative molar amounts could be determined. The level of expression of the downstream gene was 0.30 to 0.36 that of the upstream one. This determined the magnitude of RSV transcriptional polarity across a gene pair and confirmed that this value was very similar among the various intergenic regions. Minigenome transcription also yielded a CAT-LUC readthrough mRNA at a level 0.10 to 0.13 that of the LUC mRNA. In summary, the structurally diverse RSV intergenic regions do not appear to play a role in modulating RSV gene expression.