amanitins and sarkosyl

In the absence of DNA, purified yeast RNA polymerase II can bind RNA to form a binary complex. RNA in such RNA-RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA, including TFIIS-dependent cleavage and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by alpha-amanitin. Hence, by several criteria the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes in which the catalytic site for nucleotide addition is positioned at or near the 3'-OH terminus of the RNA. These findings are consistent with a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein-binding sites located up to 10 nt apart.

Topics: Amanitins; Base Sequence; Detergents; Gene Expression Regulation, Fungal; Magnesium; Models, Genetic; Molecular Sequence Data; Nucleotides; Protein Binding; RNA Polymerase II; RNA, Fungal; Sarcosine; Transcription Factors; Transcription Factors, General; Transcription, Genetic; Transcriptional Elongation Factors

Serum amyloid A (SAA) is a plasma apolipoprotein produced by the liver in response to inflammatory stimuli. The murine SAA gene family is made up of three genes, SAA1, SAA2, and SAA3, plus a pseudogene. The SAA1 and SAA2 genes are highly homologous while the SAA3 gene has diverged substantially from the other two genes. Using small fragments from the cloned genes, we have analyzed the expression of each gene in the SAA family. Within 12 h after endotoxin administration, total liver SAA mRNA increases by 2000-fold, reaching approximately 20,000 transcripts/cell. Each gene accounts for approximately one-third of total SAA mRNA transcripts at this time. The increase is specific, since the levels of the mRNAs encoding albumin and apolipoprotein A-I in liver decrease 2-fold by 24 h. This correlates with a 2-fold decrease of the serum concentrations of these two proteins as well as their in vitro protein synthesis in primary hepatocytes. SAA1+2 mRNAs maintain their maximum levels until 36 h after lipopolysaccharide administration, while SAA3 mRNA is degraded to 20% its maximal level. As assayed by in vitro transcription in isolated hepatocyte nuclei, total SAA gene transcription increases at least 300-fold during the inflammatory response. The transcription rates of the individual SAA genes are similar during the initial stages of this response, reaching peak levels at 3 h. A comparison of the rates of SAA gene transcription and SAA mRNA accumulation suggests that SAA mRNA levels are regulated during the acute phase response by increased transcription and mRNA stabilization.

Topics: Albumins; Amanitins; Amyloid; Animals; Apolipoprotein A-I; Apolipoproteins A; Endonucleases; Gene Expression Regulation; Heparin; Inflammation; Lipopolysaccharides; Mice; Nucleic Acid Hybridization; RNA, Messenger; Sarcosine; Serum Amyloid A Protein; Single-Strand Specific DNA and RNA Endonucleases; Transcription, Genetic