Primary Transcription Product Research Articles

Mapping of 5′ and 3′-ends of sunflower mitochondrial nad6 mRNAs reveals a very complex transcription pattern which includes primary transcripts lacking 5′-UTR

Nad6 orf, encoded in the sunflower mitochondrial genome in single copy contains a non-conserved 3′-extension. The transcription of the nad6 locus generates a highly complex pattern. Three main transcripts of 1240, 960 and 870 nt have been characterized by different approaches. The two smaller ones are apparently the most abundant of the steady-state RNA population and are generated as primary transcription products as well as by processing of the 1240 nt transcript. Their 5′-UTRs are absent or very short. Whereas the 3′-ends of the 960 nt transcripts contain a TGA codon the shorter one terminates at positions excluding the stop codon. The fate of transcripts to promote the synthesis of NAD6 sunflower protein seems, thus, to rely on the occurrence of mechanisms yet to be identified.

Characterization of four ribosomal RNA operons in the genome of Agrobacterium tumefaciens MAFF301001.

The four ribosomal RNA (rrn) operons rrnA, rrnB, rrnC and were identified, sequenced completely and characterized individually rrnD in the whole genome of A tumefaciens MAFF301001. These rrn operons were located in the two topologically different chromosomal DNAs; rrnA and rrnD in the linear chromosome and rrnB and rrnC in the circular chromosome. Each operon coded for three ribosomal RNA subunit genes; 16S, 23S and 5S rRNA. The 16S-23S internal transcribed spacers (ITS) of the four rrn operons contain genes for tRNA-Ile and tRNA-Ala and tRNA-Met downstream of 5S rDNA gene while the intergenic spacer between 23S rDNA and 5S rDNA lacked tRNA genes. Sequence alignment of 23S rRNAs of A. tumefaciens MAFF301001 and C58 strains showed unrelated sequences near the 5' end region suggesting the presence of intervening sequence (IVS). Primer extension analyses revealed that the primary transcription products for 16S and 23S rRNAs are 1497 and 2877 bases long, respectively.

RU5 of Mason-Pfizer monkey virus 5' long terminal repeat enhances cytoplasmic expression of human immunodeficiency virus type 1 gag-pol and nonviral reporter RNA.

Retroviruses utilize an unspliced version of their primary transcription product as an RNA template for synthesis of viral Gag and Pol structural and enzymatic proteins. Cytoplasmic expression of the gag-pol RNA is achieved despite the lack of intron removal and the presence of a long and highly structured 5' untranslated region that inhibits efficient ribosome scanning. In this study, we have identified for the first time that the 5' long terminal repeat (LTR) of Mason-Pfizer monkey virus (MPMV) facilitates Rev/Rev-responsive element-independent expression of HIV-1 gag-pol reporter RNA. The MPMV RU5 region of the LTR is necessary and directs functional interaction with cellular posttranscriptional modulators present in human 293 and monkey COS cells but not in quail QT-6 cells and does not require any viral protein. Deletion of MPMV RU5 decreases the abundance of spliced mRNA but has little effect on cytoplasmic accumulation of unspliced gag-pol RNA despite complete elimination of detectable Gag protein production. MPMV RU5 also exerts a positive effect on the cytoplasmic expression of intronless luc RNA, and ribosomal profile analysis demonstrates that MPMV RU5 directs subcellular localization of the luc transcript to polyribosomes. Our findings have a number of similarities with those of reports on 5' terminal posttranscriptional control elements in spleen necrosis virus and human foamy virus RNA and support the model that divergent retroviruses share 5' terminal RNA elements that interact with host proteins to program retroviral RNA for productive cytoplasmic expression.

Translation is not required To generate virion precursor RNA in human immunodeficiency virus type 1-infected T cells.

The retroviral primary transcription product is a multifunctional RNA that is utilized as pre-mRNA, mRNA, and genomic RNA. The relationship between human immunodeficiency virus type 1 (HIV-1) unspliced transcripts used as mRNA for viral protein synthesis and as virion precursor RNA (vpRNA) for encapsidation remains an important question. We developed a biochemical assay to evaluate the hypothesis that prior utilization as mRNA template for protein synthesis is necessary to generate vpRNA. HIV-1-infected T cells were treated with translation inhibitors under conditions that maintain virus production. Immunoprecipitation of newly synthesized HIV-1 Gag protein revealed that de novo translation is not necessary to sustain assembly, release, or processing of Gag structural protein. Both newly synthesized protein and steady-state Gag are competent for assembly, and the extracellular accumulation of Gag is proportional to the intracellular abundance of Gag. As early as 2 h after transcription, newly synthesized RNA is detectable in cell-free virions and encapsidation is sustained upon inhibition of host cell translation. Results of both [(3)H]uridine incorporation assays and HIV-1-specific RNase protection assays (RPAs) indicate that translation inhibition reduces the absolute amounts of both cytoplasmic and virion-associated RNA. Evaluation of encapsidation efficiency by RPA revealed that the cytoplasmic availability of vpRNA is increased, indicating that HIV-1 unspliced mRNA can be rerouted to function as vpRNA. Our data contrast with results from the HIV-2 and murine leukemia virus systems and indicate that HIV-1 unspliced RNA constitutes a single functional pool that can function interchangeably as mRNA and as vpRNA.

Mutations affecting mRNA processing and fimbrial biogenesis in the Escherichia coli pap operon

The Escherichia coli pap genetic determinant includes 11 genes and encodes expression of Pap pili on the bacterial surface. An RNase E-dependent mRNA-processing event in the intercistronic papB-papA region results in the accumulation of a papA-gene-specific mRNA in considerable excess of the primary papB-papA mRNA transcription product. We have introduced mutations in the intercistronic region and studied the effect in vivo of these mutations on the processing event, PapA protein expression, and the biogenesis of fimbriae on the bacterial surface. Our studies establish that mRNA processing is an important event in the mechanism resulting in differential gene expression of the major pap operon. The deletion of sequences corresponding to the major cleavage site abolished processing, reduced expression of PapA protein, and resulted in "crew-cut" bacteria with short fimbrial structures on the bacterial surface. Only a limited part of the intercistronic region appeared to be required as the recognized target for the processing to occur. Upstream sequences to a position within 10 nucleotides of the major RNase E-dependent cleavage site could be deleted without any detectable effect on papB-papA mRNA processing, PapA protein expression, or fimbria formation. Substitution mutations of specific bases at the cleavage site by site-directed mutagenesis showed that there were alternative positions at which cleavage could be enhanced, and tests with an in vitro processing assay showed that such cleavages were also RNase E dependent. Our findings are discussed in relation to other fimbrial operons and other known targets of the RNase E endoribonuclease.

A family of non-allelic tRNAGUUVal genes from the cellular slime mold Dictyostelium discoideum

A haploid genome of the cellular slime mold Dictyostelium discoideum contains at least 14 non-allelic gene copies coding for a tRNA GUU Val. The structure, genomic organization, and expression of these genes have been analyzed in relation to stages of the developmental cycle. So far, 13 tRNA GUU Val genes have been isolated and characterized. All genes contain identical mature tRNA-coding regions, and consequently identical gene internal promoter elements. However, different genes differ with respect to their 5'- and 3'-flanking regions, although a certain degree of sequence conservation seems apparent. Different members of this tRNA gene family appear to be randomly dispersed along the seven D. discoideum chromosomes, and not clustered at any one genomic location. In vivo expression of individual genes was studied in yeast. All but one tRNA GUU Val gene are actively transcribed, though with different efficiencies. There is also evidence that not all of these tRNA genes are constitutively transcribed in Dictyostelium throughout the developmental cycle. One characteristic primary transcript can only be detected in cells of the late preaggregation phase, whereas growing cells, cells in the stationary phase or cells harvested 4 h after the onset of development do not seem to carry this transcript. This product seems to be transcribed from a gene of an unusual structure. Although this particular gene has not yet been isolated, it can be predicted from the sequence of the cDNA synthesized from primary transcription products of this putative gene, that it is composed of nt 1–54 of a 3'-truncated tRNA GUU Val gene linked to a bona fide tRNA GUU Val gene.

The size and heterogeneity of the messenger RNA associated with 70 S monosomes from down-shifted Escherichia coli

After an energy source shift-down, Escherichia coli accumulates 70 S ribosome mRNA complexes (“70 S monosomes”). The monosome mRNA strands are predominantly primary transcription products with purine nucleoside 5′-triphosphate and 5′-diphosphate termini present at a 1:2 ratio. The number-average chain length is 564 ± 30 nucleotides, indicating that the population represents primarily monocistronic mRNAs. Digestions with endonucleases and exonucleases indicate that the ribosomes lie near the 5′ ends of the mRNA strands and that the majority of the mRNA strands contain 5′-proximal “leader” sequences (average 10 nucleotides) outside the protective boundary of the ribosome. These data are consistent with the hypothesis that the increased functional stability of mRNA in down-shifted cells may result from protection by bound ribosomes of endonuclease-susceptible site(s) near the 5′ ends of the mRNA strands.

Heat-shock puff 93 D from Drosophila melanogaster: accumulation of a RNP-specific antigen associated with giant particles of possible storage function.

The monoclonal antibody P11 is directed against a 38 000 dalton protein of Drosophila melanogaster. On polytene chromosomes this protein is present in a subset of the RNA polymerase II-containing loci. Here we show by density centrifugation and enzyme-linked immunosorbent assay tests that the P11 antigen is part of nuclear ribonucleoprotein (RNP) complexes. Indirect immunofluorescence shows that, after prolonged heat-shock, the P11 antigen is present only in the heat-shock puff 93 D. Identical distribution patterns were obtained with another monoclonal antibody, Q18. Unlike P11, this antibody also cross-reacts with D. hydei and D. virilis polytene chromosomes, where the puffs 48 B and 20 CD, respectively, are the only loci prominently stained after heat-shock. The small and giant RNP complexes previously described in these puffs were also observed in puff 93 D. Both types of particle contain the P11 antigen as shown by immunoelectron microscopy. We suggest that the P11 antigen is associated with a special class of RNPs which are possibly involved in the storage of primary transcription products inside the nucleus.

Kinetics of Accumulation and Processing of Simian Virus 40 RNA inXenopus laevisOocytes Injected with Simian Virus 40 DNA

We examined the kinetics of accumulation and processing of simian virus 40 (SV40) RNA in stage 6 oocytes of Xenopus laevis microinjected intranuclearly with SV40 DNA. The rates of synthesis and degradation, cellular distribution, size, and sequence specificity of radiolabeled SV40-specific and endogenous oocyte RNA were determined. The kinetics of accumulation of SV40 RNA were biphasic, with greater than 90% of the viral RNA turning over in the nucleus with a half-life of 20 to 40 min. Although most of the primary transcription products were multigenomic in length, some stable polyadenylated SV40-specific RNA similar in size and sequence to late 19S mRNA accumulated in the cytoplasm with time. Differences in strand preference, efficiencies of transcription termination and polyadenylation, and the splice sites used in the synthesis and processing of SV40 RNA in Xenopus oocytes and monkey cells were noted. However, these differences were quantitative, rather than qualitative, in nature. Consequently, they are probably due to regulatory rather than mechanistic differences between the two cell types. We therefore conclude that Xenopus oocytes may be a useful system for studying both mechanistic and cell type-specific regulatory aspects of mRNA biogenesis from cloned DNAs. However, since only a small percentage of the initially synthesized RNA ends up in stable mRNA, it will be important to determine whether mutants of cloned DNAs that produce abnormal amounts of stable mRNAs are altered in promotion and initiation of RNA synthesis, transcription termination, RNA processing, or the stability of the resultant mRNAs.

Kinetics of accumulation and processing of simian virus 40 RNA in Xenopus laevis oocytes injected with simian virus 40 DNA.

We examined the kinetics of accumulation and processing of simian virus 40 (SV40) RNA in stage 6 oocytes of Xenopus laevis microinjected intranuclearly with SV40 DNA. The rates of synthesis and degradation, cellular distribution, size, and sequence specificity of radiolabeled SV40-specific and endogenous oocyte RNA were determined. The kinetics of accumulation of SV40 RNA were biphasic, with greater than 90% of the viral RNA turning over in the nucleus with a half-life of 20 to 40 min. Although most of the primary transcription products were multigenomic in length, some stable polyadenylated SV40-specific RNA similar in size and sequence to late 19S mRNA accumulated in the cytoplasm with time. Differences in strand preference, efficiencies of transcription termination and polyadenylation, and the splice sites used in the synthesis and processing of SV40 RNA in Xenopus oocytes and monkey cells were noted. However, these differences were quantitative, rather than qualitative, in nature. Consequently, they are probably due to regulatory rather than mechanistic differences between the two cell types. We therefore conclude that Xenopus oocytes may be a useful system for studying both mechanistic and cell type-specific regulatory aspects of mRNA biogenesis from cloned DNAs. However, since only a small percentage of the initially synthesized RNA ends up in stable mRNA, it will be important to determine whether mutants of cloned DNAs that produce abnormal amounts of stable mRNAs are altered in promotion and initiation of RNA synthesis, transcription termination, RNA processing, or the stability of the resultant mRNAs.

Nucleotide sequence of the initiation site for ribosomal RNA transcription in Drosophila melanogaster: comparison of genes with and without insertions.

The sequence of 470 nucleotides surrounding the initiation site for rRNA transcription in Drosophila melanogaster has been determined. The precise initiation site was determined first by measuring the DNA fragment protected by the rRNA precursor against digestion by the single-strand specific nuclease S1 and second by direct sequence determination of the first 13 nucleotides of the rRNA precursor. Because greater than 80% od rRNA precursor molecules have been shown previously to bear pppA or ppA 5' termini, we assume that they represent the primary transcription product. Short sequence homologies exist with the initiation regions for rRNA transcription of Xenopus laevis and Saccharomyces cerevisiae. We have determined the nucleotide sequence of the initiation region in four cloned ribosomal genes from D. melanogaster which are not interrupted and in four cloned ribosomal genes in which the 28S rRNA coding region is interrupted by a 5-kilobase type 1 insertion. Three uninterrupted genes and three interrupted genes have identical sequences in the entire analyzed region. The remaining two genes have a single identical base substitution at position -17. We have shown previously that interrupted ribosomal genes in D. melanogaster are not effectively transcribed. Because the nucleotide sequences of the region where transcription initiates are identical in genes with or without insertions, we postulate that the presence of the insertion itself may be responsible for the inactivity of the interrupted genes.

Physical map of the ribosomal ribonucleic acid gene from Tetrahymena pyriformis.

A physical map of the rRNA gene from the ciliated protozoan Tetrahymena pyriformis has been determined. The isolated rDNA palindrome was labeled by nick translation and digested with the sequence-specific endonucleases KpnI, BclI, PvuII, PstI, SstI, AvaI, AvaII, HpaI, BamHI, Bg/II, or HindIII, and the molecular weights of the products were determined by gel electrophoresis. The DNA fragments generated by digestion with each endonuclease were ordered into a unique sequence either by analyzing the products produced by concomitant cleavage with one or more additional restriction enzymes or by hybridizing to 17S and 25S rRNA. Hybridization of the 35S rRNA primary transcription product to HindIII and BamHI restriction fragments localizes the promoter to the left of 16.9% and the terminator to the region between 71.6% and 78% on the physical map.

The structure and function of tRNA genes of higher eukaryotes.

The most recent findings concerning the structure and function of tRNA genes of higher eukaryotes are discussed in an exemplary way. The tRNA genes of higher organisms are either dispersed or clustered at different sites of the genome. Clusters contain tRNA genes oriented in both directions and on both strands of the DNA with spacers of various length inbetween. Some genes contain intervening sequences close to the 3' side of the anticodon. The primary transcription product possesses a 5' leader and a 3' trailer sequence which are removed by several maturation steps in a strict temporal and spacial order. Internal transcription control regions (promotors) are located at the 5' and 3' ends of the mature tRNA coding section of the tRNA gene. External sequences modulating the efficiency of the expression are present at the immediate 5' ends of the genes. Transfer RNA genes are located nonrandomly in the nucleosomes.

Dimeric transfer RNA precursors in S. pombe.

Sequence analysis of a Schizosaccharomyces pombe DNA fragment revealed two tRNA coding regions separated by a seven nucleotide spacer. the 5'-proximal tRNA gene encodes a tRNAUCGSer sequence, which is interrupted by a 16 nucleotide intron at the 3' side of the base adjacent to the anticodon. The second tRNA gene encodes an initiator tRNAMet sequence. This DNA fragment, cloned into pBR322, was used as template for in vitro transcription in a nuclear extract of Xenopus oocytes. The tRNA genes were transcribed into one RNA precursor which contained both tRNA sequences. The primary transcription product initiates with pppG, contains a 9 nucleotide leader sequence, a 16 nucleotide intron, a 7 nucleotide spacer between the two tRNA molecules and an 8--9 nucleotide trailer sequence. RNA initiation was only observed upstream of the 5'-proximal tRNASer. We used RNA analysis to establish a sequence of the enzymatic steps of tRNA maturation in the nuclear extract. The first step in processing the dimeric precursor is an endonuclease cleavage which generates the mature 5' end of the tRNAMet. Further steps include the removal of the flanking sequences and addition of the CCAOH 3' terminus. The last step is the splicing of the tRNASer precursor to remove the intervening sequence.

In vitro splicing of the ribosomal RNA precursor in isolated nucleoli from Tetrahymena.

The macronuclear rRNA genes of Tetrahymena thermophila contain an 0.4 kb intervening sequence in the 26S rRNA coding region. The sequence is represented within the primary transcription product. We demonstrate in this paper that the enzyme activities necessary for the endonucleolytic cleavage as well as for the ligation of the transcript are associated with the isolated. The intervening sequence is excised as an unique molecule, which is stable in vitro. About 50% of the in vitro synthesized RNA is processed. Faithful in vitro transcription occurs in the presence of the divalent ions Mg2+, Mn2+ and Co2+ while processing takes place only in the presence of Mg2+. The absolute requirement for Mg2+ in the excision reaction enables us to synthesize labelled pre-rRNA in the presence of Mn2+ or Co2+. The synthesized RNA can be used as a substrate in studies of th processing enzymes in vitro.

The primary transcription product of a silkworm alanine tRNA gene: Identification of in vitro sites of initiation, termination and processing

A 13.5 Kb fragment of Bombyx mori DNA containing a single tRNA 2 Ala gene has been cloned, and transcribed in vitro with Xenopus germinal vesicle extracts. The primary transcription product of the tRNA 2 Ala gene has been isolated and shown to possess an unprocessed triphosphorylated 5′ terminus. Products resulting from processing of this transcript have also been isolated and characterized. Complete nucleotide sequence analysis of this cloned alanine tRNA gene and its primary transcript shows that transcription initiates three nucleotides away from the mature tRNA 2 Ala 5′ end and terminates in a U cluster 22 nucleotides beyond the last encoded 3′ nucleotide of the mature species. Sequence determination of the products of in vitro maturation shows that in contrast to the tRNA processing mechanism characteristic of procaryotes, the extra 3′-nucleotides in this silkworm tRNA precursor are removed by a single endonucleolytic cleavage.

The intervening sequence in the 26s rrna coding region of t. thermophila is transcribed within the largest stable precursor for rRNA

We studied the transcription of the intervening sequence in the 26S rRNA coding region of the extrachromosomal rDNA molecules in the macronucleus of T. thermophila by hybridization of purified nuclear rRNA precursors or cytoplasmic 26S rRNA to purified native rDNA or specific rDNA restriction fragments. Examination of R loop hybrids in the electron microscope and analyses of S1-protected rDNA fragments in alkaline agarose gels showed that mature 26S rRNA, nuclear pre-26S rRNA and a fraction of the pre-rRNA molecules containing both the sequences for 17S and 26S rRNA all lack the region corresponding to the intervening sequence. The rest of the pre-rRNA molecules, however, hybridize in a colinear fashion to the whole coding region, and thus must contain the intervening sequence. We can conclude from these results that the intervening sequence is transcribed within the primary transcription product of the rDNA, and that the post-transcriptional removal of the intervening RNA sequence is a very early processing event in this organism.

Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40.

Simian virus 40 deletion mutants were constructed lacking specifically the intervening sequences for a late viral mRNA. The construction method involved the replacement of portions of the late simian virus 40 genes with the DNA segment from reverse transcription of the viral mRNAs. Restriction endonuclease cleavage and sequence analysis confirmed the precise structure of the mutant DNAs and demonstrated that they contained the genetic information for VP1, including all potential 5' ends for the late viral RNAs. Thus, the primary late transcription product(s) of this mutant should have the structure of functional 16S mRNAs. Complementation analysis as well as immunoprecipitation showed, however, that deletion of the intervening sequences from this mutant prevented the expression of VP1. The nature of this failure appears to be a defect in the posttranscriptional processing of the viral RNA. These results indicate that splicing is an essential function in the biogenesis of certain mRNAs.

Lengths of transcriptional units coding for the heat shock proteins in Drosophila melanogaster

The lengths of the transcription units coding for the Drosophila melanogaster heat shock proteins were determined using the ultraviolet promoter mapping technique. The figures were in good agreement with the transcription unit lengths of three of the heat shock cytoplasmic RNA species as determined by the same technique. The rates of gene inactivation were first-order with respect to ultra-violet irradiation dose, suggesting that there are single genes of a given species in each transcriptional unit. The length of the transcription unit coding for the major heat shock protein, the 70,000 M r protein, was found to be 6.8 × 10 3 bases. The length of the transcription unit of the 2.6 × 10 3-base messenger RNA coding for the 70,000 M r protein was found to be 5.5 × 10 3 to 6.5 × 10 3 bases by two independent methods and is in good agreement with the determination for the protein. In general, the transcription units contained 3.5 to 10 times as much DNA as is needed to code for the amino acid sequence of the respective proteins. The cytoplasmic RNA transcription units were 1.5 to 6.5 times the molecular lengths of the respective RNA species. The results suggest that there is appreciable post-transcriptional cutting of the primary transcription product of these genes. The lengths of these transcription units are about one-third to one-quarter the length of DNA in an average-sized chromomere.

A small, unstable RNA molecule of Escherichia coli: spot 42 RNA : I. Nucleotide sequence analysis

We have determined the structure of an unstable, small RNA of Escherichia coli, called spot 42 RNA (Ikemura & Dahlberg, 1973 a,b). Spot 42 RNA is 109 nucleotides long, has no modified nucleotides and is very likely a primary transcription product. The nuoleotide sequence and partial digestion data support a secondary structure with at least one hairpin loop. Contained within this RNA are a possible ribosome binding site, a protein synthesis initiator A-U-G and an in-phase U-G-A terminator, giving spot 42 RNA a messenger RNA-like character. Most of the 14 triplets between the A-U-G and U-G-A are codons for hydrophobic amino acids such as leucine, phenylalanine, valine and isoleucine. The 3′-terminal sequence is -U-U-U-U-U-U-A OH which is preceded by a G + C-rich hairpin looped region. The sequence and structure of spot 42 RNA share many features with other bacterial and bacteriophage RNAs, some of which have been shown to be leader RNAs (Bertrand et al., 1976).