Potential Stem-loop Structures Research Articles

Analysis of Binding Elements in the Human Immunodeficiency Virus Type 1 Genomic RNA and Nucleocapsid Protein

We previously used RNA gel mobility shift assays to demonstrate specific binding of the HIV-1 gag precursor polyprotein and nucleocapsid (NC) protein to HIV-1 RNA and to map the binding elements in each species by mutagenesis. Here we report finer mapping of binding elements in the HIV-1 genomic RNA and NC protein, performed by analyzing the binding behavior of fragments of each species in the gel shift assay. With regard to the RNA, the strongest binding activity resided in a 120-nucleotide segment flanking the gag start codon, containing three potential stem-loop structures. Binding analysis of various combinations of these three potential stem-loop structures and their flanking sequences revealed that no one element could bind to the gag polyprotein or NC protein as well as the entire 120-nucleotide segment. Mutational analysis of the NC protein showed that two nonoverlapping regions exhibited specific binding for HIV-1 RNA. Each region includes a Cys-His box, though each box could not bind to HIV-1 RNA on its own. In contrast, a construct lacking both boxes exhibited primarily nonspecific RNA-binding activity.

Deletion Analysis of the Lambda tR1 Termination Region: Effect of Sequences near the Transcript Release Sites, and the Minimum Length of Rho-dependent Transcripts

In order to determine how much of the natural sequence is required for function of the λtR1 rho-dependent terminator, and to determine the minimum length required, we made two deletion series of a tR1 derivative that contains mostly foreign DNA, encoding C-rich RNA, substituted for the natural upstream sequences of tR1. We find the minimum transcript length to be 85 to 90 nucleotides, although an additional 10 to 25 nucleotides provide more efficient termination. Sequences as close as ten nucleotides to the release sites could be replaced without destroying termination, although termination efficiency was reduced by substitution of these proximal regions with DNA encoding the cytidine rich RNA that is active at upstream sites. These results suggest that sequences proximal and distal to release sites have different functions and optimal structures for rho activation. We also show that two potential stem-loop structures in the tR1 region are not essential for terminator function, or for pausing at the release region. The results are consistent with the model that any pause site downstream of DNA encoding unstructured C-rich RNA is a potential rho-dependent terminator.

Identification and characterization of the terminators of the lys and P transcripts of bacteriophage Mu

Transcription during the lytic cycle of phage Mu occurs in three phases: early, middle, and late. Late transcription requires the Mu C protein and initiates at four promoters: Plys, PI, PP, and Pmom. Northern blot analysis of total RNA isolated 30 min after heat induction of Mu cts lysogens demonstrated that the full-length lys and P transcripts were approximately 7.6 and 6.3 kb long, respectively. The 3' ends of the lys and P transcripts were further localized by S1 nuclease mapping to intergenic regions between G and I and between U and U' in both the G(+) and G(-) orientations of the invertible G segment, respectively. As expected, when DNA fragments containing these termination regions were cloned into plasmids between Pgal and the galK gene, they showed efficient termination activity, even in a Rho-deficient background. Deletion analysis indicated that efficient termination required the presence of potential RNA stem-loop structures immediately preceding the RNA 3' ends. For the P transcript from phage with the G(-) orientation, full termination activity required both the region containing the stem-loop structure and upstream sequences. Taken together, these results suggest that the transcription termination sites of the lys and P transcripts are Rho-independent terminators.

Structural features of the 5′ upstream regulatory region of the gene encoding rat amyloid precursor protein

The 5′ upstream regulatory region of the gene encoding the rat amyloid precursor protein (APP) was cloned and sequenced. It lacks both a TATA box and a CAAT box, has a high G + C content (68%), is 89% homologous to the corresponding region of the mouse APP gene, and 82% homologous to the corresponding region of the human APP gene. This region contains putative regulatory elements both 5′ and 3′ to the probable transcription start point ( tsp). There are consensus DNA sites for the binding of SP1, AP2, AP4 and GC factor (GCF) proteins, and two GC boxes with the consensus sequence, 5′-GGGYGCRG. Potential regulatory sites with only a single mismatch to the consensus sequences include three SP1, one API, five AP2, and two GCF sites, as well as one GC box. There are also six potential stem-loop secondary structures ( SSS) near the probable tsp. A consecutive series of elements, consisting of a GC box, AP2 site, three SSS, two SP1 sites, and AP4, API and GCF sites just upstream from the probable tsp, are well-conserved between the rat, mouse and human sequences. An additional AP2 site, two GC boxes, and two additional SSS appear to be conserved between species. However, two possible rat SP1 sites, three possible rat AP2 sites, and two possible rat GCF sites are lacking in the human. On the other hand, the rat sequence is missing four potential SP1 sites, four potential AP2 sites, and nine potential GC boxes which are found in the human sequence. Therefore, while all species contain a large variety of potential regulatory elements, the numbers and locations of these elements vary significantly from species to species. These differences may be functionally related to the appearance of amyloid plaques in older humans and Alzheimer's patients, but not in older or memory-impaired rodents.

Sequences in the 3'-untranslated region of the human cellular glutathione peroxidase gene are necessary and sufficient for selenocysteine incorporation at the UGA codon

Glutathione peroxidase (EC 1.11.1.9) is one of a unique group of prokaryotic and eukaryotic enzymes that contain the unusual amino acid selenocysteine. The genes for these selenoproteins encode for the atypical amino acid at a TGA codon (UGA in the mRNA transcripts), which normally functions as a termination signal. The present studies analyzed the functional importance of sequences in the coding and 3'-untranslated regions of transcripts of the primary human cellular glutathione peroxidase gene (GPX1) to the insertion of selenocysteine at this UGA codon. Deletions in potential stem-loop or hairpin structures in the coding region did not substantially diminish incorporation of selenocysteine into glutathione peroxidase transiently expressed by the pCMV4 vector in COS-1 cells. However, selenocysteine insertion was completely abolished by deletion of four-nucleotide sequences in the 3'-untranslated region from within a conserved "selenocysteine insertion sequence" motif also found in the 3'-untranslated region of mammalian genes for other selenoproteins. Moreover, in constructs fusing the glutathione peroxidase 3'-untranslated region to the coding region of rab5b (an unrelated protein normally without any selenium moiety), the glutathione peroxidase 3'-untranslated region was sufficient to direct the translation of an opal (UGA) mutation as selenocysteine. Thus, our data directly demonstrate the importance of the selenocysteine insertion motif in the glutathione peroxidase gene and specifically show that sequence elements in the 3'-untranslated region are both necessary and sufficient for translational insertion of selenocysteine at a UGA codon in eukaryotic mRNA.

Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis

Acetyl coenzyme A (CoA) carboxylase catalyzes the synthesis of malonyl-CoA, the first intermediate of fatty acid synthesis. The Escherichia coli enzyme is encoded by four subunits located at three different positions on the E. coli chromosome. The accBC genes lie in a small operon at min 72, whereas accA and accD are located at min 4.3 and 50, respectively. We examined the expression of the genes that encode the E. coli acetyl-CoA carboxylase subunits (accA, accBC, and accD) under a variety of growth conditions by quantitative Northern (RNA) blot analysis. We found a direct correlation between the levels of transcription of the acc genes and the rate of cellular growth. Consistent results were also obtained upon nutritional upshift and downshift experiments and upon dilution of stationary-phase cultures into fresh media. We also determined the 5' end of the accA and accD mRNAs by primer extension and did transcriptional fusion analysis of the previously reported accBC promoter. Several interesting features were found in the promoter regions of these genes, including a bent DNA sequence and an open reading frame within the unusually long leader mRNA of the accBC operon, potential stem-loop structures in the accA and accD mRNA leader regions, and a stretch of GC-rich sequences followed by AT-rich sequences common to all three promoters. In addition, both accA and accD are located in complex gene clusters. For example, the accA promoter was localized within the upstream polC gene (which encodes the DNA polymerase III catalytic subunit), suggesting that additional regulatory mechanisms exist.

Isolation, characterization, and in vitro expression of a cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase.

A cDNA that encodes the kidney isoenzyme of the mitochondrial glutaminase (pGA) was generated by recombination of two cDNAs that were isolated from a random-primed rat brain lambda gt11 library. pGA encodes 674 amino acids which includes an N-terminal sequence of 16 residues that should form an amphipathic helix, typical of a mitochondrial targeting sequence. Residues 73-90 correspond to the N-terminal sequence of the more abundant 65-kDa glutaminase peptide. In vitro transcription and translation of pGA yields a 72-kDa peptide that is immunoprecipitated with glutaminase-specific antibodies. Incubation of the glutaminase precursor with isolated mitochondria yields the 68- and 65-kDa peptides that are characteristic of the mature glutaminase. Thus, the two mature glutaminase peptides are synthesized from a single precursor. The complete 3' nontranslated region of the GA mRNA was characterized by sequencing a GA cDNA (pGA12) that was isolated from an oligo(dT)-primed rat kidney lambda gt10 library. This segment contains numerous AU-rich regions, four potential stem-loop structures, and a 48 base pair repeat of CA dinucleotides. Such domains may contribute to the increased stability of the GA mRNA that occurs in response to metabolic acidosis.

Regulation of the Salmonella typhimurium aroF gene in Escherichia coli

The Salmonella typhimurium aroF gene, encoding the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, was localized to a chromosomal PstI fragment by Southern blotting with an Escherichia coli aroF probe. This fragment was cloned by screening a plasmid library for complementation of an E. coli aroF mutant. The nucleotide sequence of S. typhimurium aroF was determined and compared with its E. coli homolog. The nucleotide sequences are 85.1% identical, and the corresponding amino acid sequences are 96.1% identical. The E. coli genes encoding the three DAHP synthase isoenzymes are evolutionarily more distant from one another than are the homologous aroF genes of E. coli and S. typhimurium. The S. typhimurium aroF regulatory region contains three imperfect palindromes, two upstream of the promoter and one overlapping the promoter, that are nearly identical to operators aroFo1, aroFo2, and TyrR box 1 of E. coli. The aroFo1 and aroFo2 sequences of the two organisms are each separated by three turns of the DNA helix with no sequence similarity. The 5' ends of the aroF transcripts for both organisms contain untranslated regions with potential stem-loop structures. Translational fusions of the aroF regulatory regions to lacZ were constructed and then introduced in single copy into the E. coli chromosome. beta-Galactosidase assays for tyrR-mediated regulation of aroF-lacZ expression revealed that the E. coli TyrR repressor apparently recognizes the operators of both organisms with about equal efficiency.

Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt

The nucleotide sequence of the gbp gene, which encodes the glucan-binding protein (GBP) of Streptococcus mutans, was determined. The reading frame for gbp was 1,689 bases. A ribosome-binding site and putative promoter preceded the start codon, and potential stem-loop structures were identified downstream from the termination codon. The deduced amino acid sequence of the GBP revealed the presence of a signal peptide of 35 amino acids. The molecular weight of the processed protein was calculated to be 59,039. Two series of repeats spanned three-quarters of the carboxy-terminal end of the protein. The repeats were 32 to 34 and 17 to 20 amino acids in length and shared partial identity within each series. The repeats were found to be homologous to sequences hypothesized to be involved in glucan binding in the GTF-I of S. downei and to sequences within the protein products encoded by gtfB and gtfC of S. mutans. The repeated sequences may represent peptide segments that are important to glucan binding and may be distributed among GBPs from other bacterial inhabitants of plaque or the oral cavity.

Sequence analyses and structural predictions of double-stranded RNA segment S1 and VP7 from United States prototype bluetongue virus serotypes 13 and 10

The nucleotide sequence of segment S1 and the deduced amino acid sequence of VP7 from bluetongue virus (BTV) serotype 13 was determined. Sequences were obtained by use of standard dideoxy DNA sequencing and by direct sequencing of genomic double-stranded RNA (dsRNA). The dsRNA was sequenced with a new dideoxy protocol that produces 300 to 350 bases per set of reactions. Segment S1 is 1156 by long and contains one long open reading frame capable of coding for 349 amino acids. The protein, VP7, is rather hydrophobic, and has a calculated molecular weight of 38,619 and a net charge of +1.5 at pH 7.0. Segment S1 of BTV-13 has 79.6% of its nucleotides conserved when compared with segment S1 of BTV-10. While most of these differences occur at the third codon position of the open reading frame, the differences between the 89-base-long, 3′ noncoding regions occur predominantly in pockets at positions 1092–1098, 1112–1114, and 1125–1129. Potential stem-loop structures encompassing the stop codon of the open reading frame are proposed for both serotypes. Comparisons of VP7 from BTV-13 and BTV-10 indicate that 93.7% of the amino acid residues are conserved, including a single lysine at position 255. Secondary structure predictions infer an eight-stranded β-barrel structure between residues 150 and 250. This putative β-barrel may serve as a target for the development of drugs to combat bluetongue disease. Comparable structures detected in the core proteins of single-stranded RNA viruses from both plants and animals suggest that these viruses and BTV had a common origin.

Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E. coli.

We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E. coli. The unmodified cDNA coding for mature bovine somatotropin does not produce significant amounts of BSt in E. coli using a pBR322-derived vector. However, a translation fusion with 16 codons from trpLE in front of BSt cDNA results in greater than 20% of total cell protein as the fusion product. Analysis of transcription by measuring the rate and integrity of the mRNA confirms that a post-transcriptional event is responsible for the poor expression of the BSt cDNA. There are two potential stem-loop structures in the 5' region of the mRNA which may interfere with translation. To study their effect on translation, lacZ fusions and oligonucleotide mutagenesis were carried out. The results demonstrate that the secondary structure involving the initiation codon blocks translation initiation. Removal of this stem-loop results in a 100-fold increase in BSt expression. However, the expression level is still low, amounting to only 0.5-1% of total cell protein. High level expression can be obtained by replacement of the beginning sequence of BSt cDNA with trpLE codons. These results suggest that in addition to the secondary structure, the nucleotide sequence or amino acid context within the beginning of BSt is incompatible with one of the steps in translation initiation.

Thermodynamic stability and statistical significance of potential stem-loop structures situated at the frameshift sites of retroviruses

RNA stem-loop structures situated just 3' to the frameshift sites of the retroviral gag-pol or gag-pro and pro-pol regions may make important contributions to frame-shifting in retroviruses. In this study, the thermodynamic stability and statistical significance of such secondary structural features relative to others in the sequence have been assessed using a newly developed method that combines calculations of the lowest free energy of formation of RNA secondary structures and the Monte Carlo simulations. Our results show that stem-loop structures situated just 3' to the frameshift sites are both highly stable and statistically significant relative to others in the gag-pol or gag-pro and pro-pol junction domains (both 300 nucleotides upstream and downstream from the possible frameshift sites are included) of Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), bovine leukemia virus (BLV), human T-cell leukemia virus type II (HTLV-II), and mouse mammary tumor virus (MMTV). No other more stable, or significant folding regions are predicted in these domains.

Molecular cloning and sequence analysis of a developmentally regulated cysteine-rich outer membrane protein from Chlamydia trachomatis

Two overlapping genomic fragments have been cloned from Chlamydia trachomatis serovar L1 DNA. Sequence determination of 2530 bp has revealed two open reading frames coding for ‘cysteine-rich’ (Cr) proteins. One of these proteins was confirmed, by analysis of the inferred amino acid sequence, as the 60-kDa Cr outer membrane protein associated with differentiation of reticulate bodies (RBs) into elementary bodies (EBs). The other smaller 15-kDa protein contained a high percentage of methionine and cysteine and may correspond to a reported smaller and co-ordinately synthesised Cr outer-membrane protein also associated with RB to EB differentiation. Sequencing showed three potential stem-loop structures within the 5', 3' and intergenic regions of the cloned fragment. Southern-blot analysis revealed that the cloned fragment is conserved in ten serovars of C. trachomatis and that a strongly cross-hybridising fragment is also present in Chlamydia psittaci.

Plasmid deletion formation in Bacillus subtilis.

Plasmid pGP1 carrying a penP-lacZ fusion was used to study structural plasmid instability in Bacillus subtilis. In only one of 28 sequenced deletion junction points in the penP-lacZ region short direct repeats (10 bp) and flanking imperfect inverted repeats (12 bp) were associated with endpoints. In 27 deletions no repeated sequences of more than 3 bp were present at the endpoints. In 15 of these the sequence 5'-T-G-T-A-3' was found within 10 bp from the left endpoint. At the left cleavage sites the sequence 5'-T-T-T-3', or the 5'-A-A-A-3' complement thereof, was frequently observed. Most of the left deletion endpoints were located in potential stem-loop structures in the penP transcription/translation regulatory region, which is very rich in hyphenated dyad symmetry. Near the right deletion endpoints a sequence consisting of four G/C residues, followed by three or four A/T residues, was found in 15 cases. It is speculated that DNA topoisomerase I is involved in the formation of the deletions studied.

Stability of RNA stem-loop structure and distribution of non-random structure in the human immunodeficiency virus (HIV-I).

The stability of potential RNA stem-loop structures in human immunodeficiency virus isolates, HTLV-III and ARV, has been calculated, and the relevance to the local significant secondary structures in the sequence has been tested statistically using a Monte Carlo simulation method. Potentially significant structures exist in the 5'non-coding region, the boundary regions between the protein coding frames, and the 3' non-coding region. The locally optimal secondary structure occurring in the 5' terminal region has been assessed using different overlapping segment sizes and the Monte Carlo method. The results show that the most favorable structure for the 5' mRNA leader sequence of HIV has two stem-loops folded at nucleotides 5-104 in the R region (stem-loop I, 5-54 and stem-loop II, 58-104). A large fluctuation of segment score of the local optimal secondary structure also occurs in the boundary between the exterior glycosylated protein or outer membrane protein and transmembrane protein coding region. This finding is surprising since no RNA signals or RNA processing are expected to occur at this site. In addition, regions of the genome predicted to have significantly more open structure at the RNA level correlate closely with hypervariable sites found in these viral genomes. The possible importance of local secondary structure to the biological function of the human immunodeficiency virus genome is discussed.

The rat alpha-fetoprotein and albumin genes. Transcriptional control and comparison of the sequence organization and promoter region.

Functional and structural approaches were used to characterize the transcription units of the rat alpha-feto-protein (AFP) and albumin genes. A cell-free nuclear transcription assay and several genomic clones were used to show that: 1) the rate of transcription of these genes is closely related to the levels of corresponding mRNAs in the yolk sac and during rat liver development, indicating that the expression of the albumin and AFP genes is mainly regulated at the transcriptional level in the rat, and 2) the in vivo 5' end boundaries of the rat AFP and albumin transcription domains were mapped near the respective first exons. Due to the presence of repeated sequences, the 3' end boundary of both genes could not be accurately defined in the same manner. 3) No transcription could be detected until 7 kilobases upstream from the cap site of these genes. In addition, the organization of the rat AFP gene was analyzed by restriction endonuclease mapping, S1 nuclease mapping, and nucleotide sequencing. Our results indicate that: 1) the rat AFP gene is 20 kilobase pairs long and is split into 15 exons by 14 intervening sequences; 2) the transcription initiation site of the rat AFP gene is heterogenous; 3) the 5'-flanking region upstream from the rat AFP gene exhibits 60-90% similarity with the mouse and human AFP genes while no major nucleotide identity is found with the rat albumin gene; 4) a 90-base pair sequence present as one copy upstream from the rat and mouse AFP genes is present as two copies in the human genome; 5) several inverted repeats are mapped in the 5'-flanking region indicating potential stem-loop structures. One highly conserved structure encompasses an enhancer-like core sequence and the sequence recognized by the TGGCA-binding protein.

Characterization of human DNA sequences synthesized at the onset of S-phase.

We developed a method of enrichment for DNA replicated at the onset of S-phase in synchronized human HL60 cells. About 200 such sequences were cloned. The analysis of this selected DNA sample showed that: 1) the cloned DNA fragments derive from a limited number (750-1500) of replicons; 2) there is no extensive homology between different DNA fragments; 3) they are not significantly enriched in highly repeated sequences; 4) they are enriched in snap-back (Cot = o) DNA. The sequence of the longest fragment revealed the presence of numerous signals collected in a few hundred nucleotides: 1) homology with the origin of replication of human Papovaviruses usually associated with potential stem-loop structures; 2) binding sites for known transcription factors and for another nuclear factor; 3) potential binding sites for the chromosome "scaffold".