Biomaterial Database

Analysis of RNA chain elongation and termination by Saccharomyces cerevisiae RNA polymerase III. 1994

H Matsuzaki, and G A Kassavetis, and E P Geiduschek

Department of Biology, University of California, San Diego, La Jolla 92093-0634.

When Saccharomyces cerevisiae RNA polymerase (Pol) III transcribes the S. cerevisiae SUP4 tRNA(Tyr) gene, it is obliged to navigate past a large, multi-subunit DNA-bound complex of proteins. We have analyzed individual steps of RNA chain elongation on this gene. Slow steps of transcriptional initiation were by-passed by forming 5'-end-labeled, arrested and precisely positioned transcription complexes. Synchronous resumption of chain elongation by these complexes allowed a single round of RNA synthesis and termination to be analyzed in detail. Results for synthesis at 20 degrees C and 0 degrees C, in the presence of 100 microM and 1 mM ribonucleoside triphosphates (NTPs) are presented. RNA chain elongation through assembled transcription complexes was uneven but relatively rapid: at 20 degrees C with 1 mM NTPs, the fastest RNA chains elongated at an average rate of 29 nucleotides (nt)/second, and the median RNA chains elongated at 21 to 22 nt/second on average. These rates are comparable with a recent measurement of the average rate of chain elongation in vivo by Drosophila RNA polymerase II at 25 degrees C. At 0 degree C, RNA chain elongation rates were, on average, approximately 30-fold slower. Quantitative analysis of the individual steps of RNA chain elongation showed that steps of adding U and A to U-terminated RNA chains tended to be relatively slow, and to be more strongly influenced by nucleotide concentration. Termination of transcription occurred in the sequence T7GT6 (in the non-template DNA strand) and was progressive. Transcripts with five, six and seven U residues were formed, and there was even slow readthrough of the T7 stretch, with GU3 adding rapidly, suggesting that incorporation of a single G into the RNA chain served to reset elongation rates substantially or entirely. Stripping transcription factor (TF) IIIC from transcription complexes did not substantially increase overall RNA chain growth rate, but did diminish pausing at a single site upstream of the boxB binding site of TFIIIC. The TFIIIC-generated delay at this single site was estimated to be only approximately 0.15 to 0.2 seconds at 20 degrees C. Quantitative analysis of RNA chain elongation yielded kinetic parameters for the individual steps of nucleotide addition that were used in computer simulations of RNA chain growth. Elongation modeled as a simple sequence of pseudo-first-order reactions yielded computed RNA chain length distributions that remained relatively synchronous during elongation, while observed chain growth quickly became desynchronized.(ABSTRACT TRUNCATED AT 400 WORDS)

UI	MeSH Term	Description	Entries
D008957	Models, Genetic	Theoretical representations that simulate the behavior or activity of genetic processes or phenomena. They include the use of mathematical equations, computers, and other electronic equipment.	Genetic Models,Genetic Model,Model, Genetic
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D003198	Computer Simulation	Computer-based representation of physical systems and phenomena such as chemical processes.	Computational Modeling,Computational Modelling,Computer Models,In silico Modeling,In silico Models,In silico Simulation,Models, Computer,Computerized Models,Computer Model,Computer Simulations,Computerized Model,In silico Model,Model, Computer,Model, Computerized,Model, In silico,Modeling, Computational,Modeling, In silico,Modelling, Computational,Simulation, Computer,Simulation, In silico,Simulations, Computer
D004271	DNA, Fungal	Deoxyribonucleic acid that makes up the genetic material of fungi.	Fungal DNA
D005800	Genes, Fungal	The functional hereditary units of FUNGI.	Fungal Genes,Fungal Gene,Gene, Fungal
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012320	RNA Polymerase III	A DNA-dependent RNA polymerase present in bacterial, plant, and animal cells. It functions in the nucleoplasmic structure where it transcribes DNA into RNA. It has specific requirements for cations and salt and has shown an intermediate sensitivity to alpha-amanitin in comparison to RNA polymerase I and II.	DNA-Dependent RNA Polymerase III,RNA Polymerase C,DNA Dependent RNA Polymerase III,Polymerase C, RNA,Polymerase III, RNA
D012331	RNA, Fungal	Ribonucleic acid in fungi having regulatory and catalytic roles as well as involvement in protein synthesis.	Fungal RNA
D012333	RNA, Messenger	RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.	Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated