Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01350 (gastrin)
 9,683 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe an in vitro system that emulates the specific and efficient transcriptional termination associated with the human gastrin gene terminator in vivo. The system involves a dC-tailed DNA template containing the gastrin gene terminator sequence, purified RNA polymerase II, and purified elongation factor TFIIS. In this system, the basal level of termination by RNA polymerase II at the gastrin gene terminator is specifically enhanced by netropsin, an (A + T)-rich minor groove-binding peptide. This enhanced termination is maintained even with TFIIS, which normally suppresses termination at this site. In vitro termination is terminator sequence-specific. Mutant sequences that reduce or abolish termination in vivo show corresponding reductions in activity in the in vitro system. This in vitro emulation of in vivo activities of wild-type and mutant terminators strongly suggests that netropsin and a putative termination factor may share some aspects of their biochemical mechanisms. The general applicability of this system to the study of RNA polymerase II elongation and termination is suggested by the enhancement of termination seen at both the gastrin and human histone H3.3 gene terminators.
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PMID:Netropsin specifically enhances RNA polymerase II termination at terminator sites in vitro. 131 32

Recently, we identified a specific DNA sequence from the gastrin gene that regulates RNA polymerase II transcription termination in vivo. In the studies presented here, we examined the processing and termination activity of this sequence in vitro. When present in an in vitro synthesized RNA, this sequence (U9A2U5AU4AU4AU5) does not serve as an RNA processing signal on incubation with HeLa whole-cell extract. However, transcription of template DNA in HeLa whole-cell extract does terminate near the 5' end of this sequence. Nuclease S1 and exonuclease VII mapping of the 3' region of the in vitro synthesized RNAs confirm these results. The termination activity of the sequence A9T2A5TA4TA4TA5 is independent of the distance from the promoter and of the nature of the DNA template (linear vs. circular). The termination activity of the sequence shows a strong orientation dependence. These results strongly suggest that the termination activity of this cis-acting element is modulated by a trans-acting cellular factor. The unique structural feature of this sequence, a 10.5-base-pair inverted repeat, may determine the specificity of interaction of the trans-acting factor with the cis-acting element, resulting in accurate termination of transcription.
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PMID:RNA polymerase II transcription terminates at a specific DNA sequence in a HeLa cell-free reaction. 242 12

To identify the transcription termination elements in the mouse gastrin gene, we examined RNA transcripts after stable transfection of gastrin expression plasmids into the NIH3T3 cell line. The GT-repeat region at the 3'-flanking sequence of the mouse gastrin gene acted as a transcription terminator. When the GT-repeat unit was deleted from its site, the effect of termination disappeared. Further experiment, using serial deletion mutants, revealed that the 56-38 nucleotide upstream region from the GT-repeat unit also participated in transcription termination. We propose that the upstream region of the GT-repeat unit might be recognized as a pause site by the RNA polymerase II, and an abnormal DNA structure, derived from the GT-repeat unit, might function as a blockage of polymerase processivity.
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PMID:Sequences responsible for transcription termination of the mouse gastrin gene. 749 58

Replication of human immunodeficiency virus requires Tat protein which activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex (P-TEFb). Tat binds directly through its transactivation domain to the CycT1 subunit of the P-TEFb and induces loop sequence specific binding of the P-TEFb onto nascent HIV-1 TAR RNA. By using a gel electrophoresis method and a comprehensive set of TAR loop mutants, we have identified the sequence and structural determinants for high-affinity CycT1-Tat-TAR ternary complex formation. Our results show that CycT1 and Tat binding to TAR RNA is highly cooperative, and a capacity of 85%, a Hill coefficient of 2.7, and a dissociation constant (K(D)) of 2.45 nM were observed. These results indicate that there are three binding sites on TAR RNA. CycT1 does not bind TAR RNA in the absence of Tat, and Tat binding to TAR, while detectable, is very inefficient in the absence of CycT1. It is conceivable that the CycT1-Tat heterodimer directly binds to TAR RNA in the U-rich RNA bulge region and this binding facilitates the interactions of the CycT1-Tat heterodimer at the other two sites in the RNA loop region. On the basis of our results, we suggest a model where CycT1 interacts with Tat protein and positions the protein complex to make contacts with the G34 region of the loop sequence; G34 is critical for CycT1-Tat binding and forms a C30.G34 base pair. Two functional groups, O6 and N7, at nucleotide positions 32 and 34 in the TAR loop are essential for CycT1-Tat interactions with TAR RNA. The identity of two nucleotides, U31 and G33, is not critical, but they contribute to the stabilization of the RNA-protein complex. The presence of a single-nucleotide bulge of A35 or C35 is essential for distortion of the backbone RNA structure as well as the accessibility of functional groups in the major groove of the double-helical region. CycT1-Tat interaction with TAR RNA represents another example of the flexibility and complexity of RNA structure involved in protein recognition.
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PMID:Specific HIV-1 TAR RNA loop sequence and functional groups are required for human cyclin T1-Tat-TAR ternary complex formation. 1200 1