Biomedical subjects

A Krol

Publications and source records attributed to A Krol.

At least 109 records · Page 6Linked to original sources

PubMed1988

Changing the RNA polymerase specificity of U snRNA gene promoters.

The promoter of a Xenopus tropicalis U6 gene can be transcribed by both RNA polymerases II and III. Two distinct elements, a TATA-like sequence and the region of transcription initiation, are only required for transcription by RNA polymerase III, while further common elements are required for transcription by both polymerases. Based on the unusually stringent requirement for a purine at the normal position of polymerase III transcription initiation and on the properties of mutants in this region, we suggest that RNA polymerase III itself may recognize the site of transcription initiation and thus be directly involved in efficient promoter selection. We have used the information obtained on U6 promoter structure to manufacture a U6 promoter that is RNA polymerase II-specific and to change the Xenopus U2 gene promoter specificity from RNA polymerase II to RNA polymerase III.

Animals↗

PubMed1987

A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II.

The structure of a Xenopus U6 gene promoter has been investigated. Three regions in the 5'-flanking sequences of the gene that are important for U6 expression are defined. Deletion of the first, between positions -156 and -280 relative to the site of transcription initiation, reduces transcription to roughly 5% of its original level. Deletion of the second, between -60 and -77, abolishes transcription. These regions contain not only functional but also sequence homology to the previously defined distal and proximal sequence elements (DSE and PSE) of the Xenopus U2 promoter, although U2 is transcribed by RNA polymerase II and U6 by RNA polymerase III. Competition experiments show that at least the distal sequence elements of the two promoters bind to a common factor both in vivo and in vitro. Part of the sequence recognized by this factor is the octamer motif (ATG-CAAAT). A sequence similar to the common RNA polymerase II TATA box is also shown to have an effect, albeit minor, on U6 transcription. The U6 coding region contains a good match to the A box, part of all previously characterized RNA polymerase III promoters. Deletion of this region has no apparent effect on the efficiency or accuracy of U6 transcription.

Animals↗

PubMed1987

Xenopus tropicalis U6 snRNA genes transcribed by Pol III contain the upstream promoter elements used by Pol II dependent U snRNA genes.

We have cloned and sequenced a 977bp DNA fragment, pXTU6-2, that represents the transcription unit for a Xenopus tropicalis U6 RNA gene. This basic repeating unit is reiterated ca.500-fold per haploid genome. Oocyte injections of pXTU6-2 led to the transcription of a mature-sized U6 RNA that, however, lacked internal 2'-O-methylations. These posttranscriptional modifications of U6 RNA might be cytoplasmic and could require its association with U4 RNA to be accomplished. The low alpha- amanitin sensitivity of U6 RNA synthesis in oocytes suggested that U6 RNA is transcribed by RNA polymerase III, consistent with features of the U6 RNA molecule which also contains a Box A- like intragenic control region. Inspection of X. tropicalis, mouse and human U6 DNA upstream sequences revealed the presence of a TATA box as well as of the proximal and enhancer (octamer motif) elements contained in snRNA genes transcribed by RNA polymerase II. We propose that U6 RNAs are synthesized by a specialized transcription complex consisting of RNA polymerase III and transcription factors, some of which are very likely shared with RNA polymerase II promoters.

Animals↗

PubMed1985

The two embryonic U1 RNA genes of Xenopus laevis have both common and gene-specific transcription signals.

We have cloned and sequenced the 1842-bp repeat DNA encoding the two Xenopus laevis embryonic U1 RNAs, xU1a and xU1b. Although these two U1 RNAs are almost identical in sequence and are coordinately expressed during early embryogenesis, the flanking sequences of their genes show very little homology. Both genes contain two short conserved sequences, centered around positions -55 and +19, that probably are essential for 5' and 3' end formation of U1 RNAs, respectively. Efficient transcription of either gene in stage VI oocytes requires gene-specific promoter elements, located upstream of position -220. In the xU1b gene, these required 5'-flanking sequences include an 18-bp palindrome that has potential for Z-DNA formation. When injected separately into stage VI oocytes, the xU1a and xU1b genes are equally well transcribed, but co-injection of the two genes, either as the full length repeat or as two separate subclones, results in preferential accumulation of xU1b RNA. This competitive advantage of the xU1b gene in injected oocytes apparently is the result of preferred binding of one or more transcription factors that are limiting in these oocytes.

Animals↗

PubMed1984

A family of small nucleoplasmic RNAs with common structural features.

The four small nucleoplasmic RNAs designated as U1, U2, U4 and U5 RNA have a common structural domain or domain A. It is characterized by the presence of consensus sequence Pu-A-(U)5-G-Pup in a free single-stranded region and of the sequence Py-N-Py-Gp in the top loop of a hairpin located at the 3' end of the free region. Domain A is likely to be involved in a function common to the four small RNAs. Other domains possibly involved in the specific function of U1 and U5 RNAs are also presented. Several molecular forms of a same U RNA may be found in a same cellular type. These variant U RNAs are particularly numerous for U5 RNA. The possible origin of such new molecular forms is discussed in relation to multigene families for U RNAs. U1, U2, U4 and U5 RNAs are capped (with trimethylguanosine) and the consensus sequence for their 6 first nucleotides is A-(Py)5p. Thus, their 5' end resembles that of premessenger RNA, also capped (with monomethylguanosine) and bearing A-(Py)5p. Both classes of RNA are synthesized by RNA polymerase B, but the presence of a "TATA box" upstream of the capsite has not yet been demonstrated for U RNA genes. U1, U2, U4 and U5 RNAs bear a sequence C-Py-Pu at their 3' end or very close to it. This trinucleotide might be part of a site of post-transcriptional cleavage of U RNA precursors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

U1, U2 and U5 small nuclear RNAs are found in plants cells. Complete nucleotide sequence of the U5 RNA family from pea nuclei.

U1, U2 and U5 RNAs were isolated from pea nuclei with antibody specific for 2,2,7-trimethylguanosine. The nucleotide sequence of the 3'-terminal halves of pea U1 and U2 snRNAs and the complete sequence of five out of the six U5 RNA variants isolated is given. The high number of U5 variants suggest they are encoded by a multigene family containing at least six different genes. Similar secondary structures could be derived for all of the pea U5 RNAs and although the degree of sequence conservation between plant and vertebrate U5 RNAs is as low as 35%, nearly identical secondary structures can be proposed for both RNA groups. All the snRNA species U1, U2 and U5 from pea share a structural domain, the so-called domain A, which is also common to all animal snRNA U1, U2, U4 and U5. Furthermore, a block of 22 consecutive nucleotides is conserved among pea and vertebrate U5 RNAs, from which 11 nucleotides constitute a hairpin-loop with a high number of posttranscriptional modifications. We propose that conservation of this hairpin-loop, together with domain A, is of prime importance for the functioning of U5 RNAs in plant and animal cells.

Base Sequence↗

PubMed1983

High evolutionary conservation of the secondary structure and of certain nucleotide sequences of U5 RNA.

The nucleotide sequence of chicken, pheasant, duck and Tetrahymena pyriformis U5 RNAs as well as that of new mammalian variant U5 RNAs was determined and compared to that of rat and HeLa cells U5 RNAs. Primary structure conservation is about 95% between rat and human cells, 82% between mammals and birds and 57% between the Protozoan and mammals. The same model of secondary structure, a free single-stranded region flanked by two hairpins can be constructed from all RNAs and is identical to the model previously proposed for mammalian U5 RNA on an experimental basis (1). Thus, this model is confirmed and is likely to be that of an ancestor U5 RNA. The 3' region of the U5 RNA molecule constitutes domain A, and is common to U1, U2, U4 and U5 RNAs (2). The characteristic nucleotide sequences of domain A are highly conserved throughout the phylogenetic evolution of U5 RNA suggesting that they are important elements in the function of the four small RNAs. Another region of high evolutionary conservation is the top part of the 5' side hairpin whose conserved sequence is specific to U5 RNA. It might participate in the particular function of U5 RNA.

Animals↗

PubMed1982

Accessibility of U1 RNA to base pairing with a single-stranded DNA fragment mimicking the intron extremities at the splice junction.

A DNA fragment containing a 16 nucleotide sequence mimicking the intron extremities of premessenger RNA aligned as proposed previously (1,2) in a model of splicing mechanism was prepared and used as a probe for accessibility of the 5' extremity of U1 RNA. Hybridization of U1 RNA to the probe under non denaturing conditions and digestion of the hybrid with RNase H revealed that the sequence of U1 RNA which is complementary to the extremities of introns is accessible to hybridization and to enzymes. Therefore, the configuration of isolated U1 RNA satisfies the criteria required for the alignment of introns and further enzymatic reactions of splicing.

Base Composition↗

PubMed1982

U2 RNA shares a structural domain with U1, U4, and U5 RNAs.

We previously reported common structural features within the 3'-terminal regions of U1, U4, and U5 RNAs. To check whether these features also exist in U2 RNA, the primary and secondary structures of the 3'-terminal regions of chicken, pheasant, and rat U2 RNAs were examined. Whereas no difference was observed between pheasant and chicken, the chicken and rat sequences were only 82.5% homologous. Such divergence allowed us to propose a unique model of secondary structure based on maximum base-pairing and secondary structure conservation. The same model was obtained from the results of limited digestion of U2 RNA with various nucleases. Comparison of this structure with those of U1, U4, and U5 RNAs shows that the four RNAs share a common structure designated as domain A, and consisting of a free single-stranded region with the sequence Pu-A-(U)n-G-Pup flanked by two hairpins. The hairpin on the 3' side is very stable and has the sequence Py-N-Py-Gp in the loop. The presence of this common domain is discussed in connection with relationships among U RNAs and common protein binding sites.

Animals↗

PubMed1981

The primary and secondary structure of yeast 26S rRNA.

We present the sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis as inferred from the gene sequence. The molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence. Starting from the recently proposed structure of E. coli 23S rRNA (see ref. 25) we constructed a secondary structure model for yeast 26S rRNA. This structure is composed of 7 domains closed by long-range base pairings as n the bacterial counterpart. Most domains show considerable conservation of the overall structure; unpaired regions show extended sequence homology and the base-paired regions contain many compensating base pair changes. The extra length of the yeast molecule is due to a number of insertions in most of the domains, particularly in domain II. Domain VI, which is extremely conserved, is probably part of the ribosomal A site. alpha-Sarcin, which apparently inhibits the EF-1 dependent binding of aminoacyl-tRNA, causes a cleavage between position 3025 and 3026 in a conserved loop structure, just outside domain VI. Nearly all of the located methyl groups, like in E. coli, are present in domain II, V and VI and clustered to a certain extent mainly in regions with a strongly conserved primary structure. The only three methyl groups of 26S rRNA which are introduced relatively late during the processing are found in single stranded loops in domain VI very close to positions which have been shown in E. coli 23S rRNA to be at the interface of the ribosome.

Base Sequence↗

PubMed1981

Primary and secondary structures of Escherichia coli MRE 600 23S ribosomal RNA. Comparison with models of secondary structure for maize chloroplast 23S rRNA and for large portions of mouse and human 16S mitochondrial rRNAs.

We determined 90% of the primary structure of E.coli MRE 600 23S rRNA by applying the sequencing gel technique to products of T1, S1, A and Naja oxiana nuclease digestion. Eight cistron heterogeneities were detected, as well as 16 differences with the published sequence of a 23S rRNA gene of an E.coli K12 strain. The positions of 13 post-transcriptionally modified nucleotides and of single-stranded, double-stranded and subunit surface regions of E.coli 23S rRNA were identified. Using these experimental results and by comparing the sequences of E.coli 23S rRNA, maize chloro. 23S rRNA and mouse and human mit 16S rRNAs, we built models of secondary structure for the two 23S rRNAs and for large portions of the two mit rRNAs. The structures proposed for maize chloroplast and E.coli 23S rRNAs are very similar, consisting of 7 domains closed by long-range base-pairings. In the mitochondrial 16S rRNAs, 3 of these domains are strongly reduced in size and have a very different primary structure compared to those of the 23S rRNAs. These domains were previously found to constitute a compact area in the E.coli 50S subunits. The conserved domains do not belong to this area and contain almost all the modified nucleotides. The most highly conserved domain, 2042-2625, is probably part of the ribosomal A site. Finally, our study strongly suggests that in cytoplasmic ribosomes the 3'-end of 5.8S rRNA is basepaired with the 5'-end of 26S rRNA. This confirms the idea that 5.8S RNA is the counterpart of the 5'-terminal region of prokaryotic 23S rRNA.

Animals↗

PubMed1981

Primary and secondary structures of chicken, rat and man nuclear U4 RNAs. Homologies with U1 and U5 RNAs.

U4 RNA from chicken, rat and man was examined for nucleotide sequence and secondary structure. Three molecular species, U4A, U4B and U4C were detected in the three animal species. U4A is 146 nucleotide long and U4B RNA only lacks the 3' terminal G. four nucleotides are missing at the 3'-end of U4C RNA which, in addition, differs from U4A and U4B RNAs at two internal positions. Thus, U4C RNA is encoded by another gene as U4A and U4B RNAs. Only one nucleotide substitution occurred between chicken and man showing that U4A, U4B and U4C RNAs have been extremely conserved throughout evolution. The three molecular species are capped, they contain three psi, a 2'-P methyl A and a m6A. An additional post-transcriptional modification close to the cap structure is observed in man. On the basis on an experimental study, two models of secondary structure may be proposed for U4 RNA. The 3'domain is the same in both models and is homologous to that of U1 and U5 RNAs. It consists of a single-stranded region, containing the sequence Py-(A)2-(U)n-Gp flanked by two stable hairpins probably involved in tertiary interactions. The 5' domain is less stable than the 3' domain and its structure is different in the two models. However, a long single-stranded pyrimidine region containing modified nucleotides is found in both models as in U1 and U5 RNAs. Several other nucleotide sequence homologies related to specific features of secondary structure suggest that U1, U4 and U5 RNAs derive from a common ancestor and may have common function.

Animals↗

PubMed1981

Small RNAs in HnRNP fibrils and their possible function in splicing.

Several arguments are in favor of a function of snRNA in the processing of premessenger RNA. A large fraction of snRNA is localized in hnRNP which are assumed to be the site of processing. The different snRNA species are not bound to hnRNP in a unique manner but are associated with both proteins and hnRNA which suggests the possibility of metabolic exchanges in the course of processing. There is approximately 1-2 molecules of snRNA per individual hnRNP. We reexamined the possibility that U1A RNA might serve for the alignment of the extremities of the intron sequences of premessenger RNA insuring correct condition for cutting and splicing. We found that only a UCCA (3' leads to 5') sequence at position 8-11 of U1A RNA was complementary to an AG-GU (5' leads to 3') around a putative splice point for 69 different introns sequenced so far. On the basis of secondary structure of U1A RNA, the UCCA sequence would be available for hybridization. The UCCA sequence is also present in U2 RNA and 4.5 S RNAI. It might associate with AG-GU in a manner similar to that of codon-anticodon, the stability of the complex being insured by the configuration of hnRNP. The possible formation of larger hybrids stable by themselves is unlikely upon examination of the nucleotide sequence of various introns adjacent to the splice point. As there is no direct experimental evidence for the function of snRNA in splicing, there considerations are speculative at the present time. The possibility that adenovirus encoded VA RNA would play a role in splicing was also examined. Various arguments suggest that this possibility is rather remote.

Adenoviridae↗

PubMed1981

The nuclear 5S RNAs from chicken, rat and man. U5 RNAs are encoded by multiple genes.

Preparations of chicken, rat and human nuclear 5S RNA contain two sets of molecules. The set with the lowest electrophoretic mobility (5Sa) contains RNAs identical or closely related to ribosomal 5S RNA from the corresponding animal species. In HeLa cells and rat brain, we only detected an RNA identical to the ribosomal 5S RNA. In hen brain and liver, we found other species differing by a limited number of substitutions. The results suggest that mutated 5S genes may be expressed differently according to the cell type. The set with the highest mobility corresponds to U5 RNA. In both rat brain and HeLa cells, U5 RNA was found to be composed of 4 and 5 different molecules respectively (U5A, U5B1-4) differing by a small number of substitutions or insertions. In hen brain, no U5B was detected but U5A' differing from U5A by the absence of the 3'-terminal adenosine. All the U5 RNAs contain the same set of modified nucleotides. They also have the same secondary structure which consists of two hairpins joined together by a 17 nucleotide long single-stranded region. The 3' half of the molecule has a compact conformation. Together, the results suggest that U5 RNAs are transcribed from a multigene family and that mutated genes may be expressed as far as secondary structure is conserved. The conformation of U5 RNA is likely to be related to its function and it is of interest to mention that several similarities of structure are found between U5 and U1A RNA.

Animals↗

PubMed1981

The conformation of chicken, rat and human U1A RNAs in solution.

Chicken, rat and human U1A RNAs in solution, were examined for secondary structure, using several methods including hydrolysis by various nucleases, hybridization to DNA oligomers and analysis of fragment interactions. The experimental results showed that the three U1A RNAs have the same structure, stable over a wide range of pH and ionic conditions. They allowed the selection of one out of several possible models constructed from the data of primary structure. This model is characterized by 4 hairpins and two single-stranded regions, the two hairpins from the 3' part of the molecule bearing very stable stems. In addition, the experimental results showed that in contrast to the 5' half of the molecule, the 3' half has a compact conformation probably stabilized by tertiary interactions. The 5' end of U1A RNA is accessible and free of base-pairing so that it might base-pair with regions of other RNA molecules, for instance, with the extremities of introns as has been recently proposed in a model of splicing.

Animals↗

PubMed1981

The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA.

An heterologous complex was formed between E. coli protein L1 and P. vulgaris 23S RNA. We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex. We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively. By comparison of these results with those previously obtained with the homologous regions of E. coli and B. stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA. Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E. coli 23s RNS region preceding the L1 binding site. The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA. The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor. One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA.

Animals↗

PubMed1980

Characterization of the Escherichia coli 23S Ribosomal RNA region associated with ribosomal protein L1. Evidence for homologies with the 5'-end region of the L11 operon.

The results previously obtained upon studying the L1-23S RNA complex by the fingerprint technique have been reexamined in the light of new data on 23S RNA primary structure. The 23S RNA region that remains associated with the L1 ribosomal protein after RNase digestion of the synthetic complex lies between nucleotides 2067 and 2235 from the 5'-end of the molecule. This region contains a m7G near to the 5'-end and possesses a high degree of mutability in E. coli. Three different sequences were observed in E. coli MRE 600. All three sequences differ in two positions relative to the corresponding sequence in rrnB cistron from E. coli K12. Striking homology is observed between the 23S RNA region associated with protein L1 and the 5'-part of L11 operon. This observation supports the model of feedback regulation of r-proteins synthesis proposed by Yates et al. (PNAS, 77, 1837) and strongly suggests that the region of 23S RNA located between positions 2155 and 2202 is essential for the binding of protein L1.

Bacterial Proteins↗

PubMed1980

Nucleotide sequences of nuclear U1A RNAs from chicken, rat and man.

The methods of enzymatic and chemical treatment of end-labeled RNA were applied to the determination of the nucleotide sequence of chicken and man U1A RNA and to the reexamination of that of rat U1A RNA. The chemical method allowed the easy demonstration of the cap structure. All three RNA were 165 nucleotide long. Two hitherto non described modified pyrimidines were detected close to the 5' end. Only 9 base substitutions were observed from chicken to man indicating high degree of conservation of U1A RNA through evolution.

Animals↗