Transcription and in vitro processing of yeast 5 S rRNA.

A method is described for the isolation of a yeast chromatin fraction highly enriched in ribosomal DNA sequences. In the presence of exogenous yeast RNA polymerase III, this purified chromatin actively synthesizes a set of 5 S ribosomal RNAs all of which have 5'-sequences identical with mature 5 S RNA but which end with a variable number (up to 10) of additional residues at the 3'-terminus. These extra nucleotides are precisely removed by a processing nuclease found in the chromatin supernatant fraction.

Quantification of influenza virus messenger RNAs.

The amounts of specific influenza virus cRNAs present on polyribosomes of cells treated with cycloheximide at various times after infection were measured by quantitative hybridization with 125I-labelled virus RNA segments. All species of cRNA were found associated with polyribosomes at every time point analysed. The relative abundance of the specific RNAs was in the same order as the reported relative synthesis of the influenza virus proteins. However, the concentrations of the cRNAs spanned a considerably smaller range than published protein synthetic rates, suggesting that both transcriptional and translational controls operate to regulate the final levels of influenza virus polypeptide synthesis.

Specific gene transcription in yeast nuclei and chromatin by added homologous RNA polymerases I and II.

When treated at pH less than 4.5, yeast nuclei or chromatin lose endogenous RNA synthetic activity. This activity is regained by addition of exogenous RNA polymerases. The specificity of transcription in this system by homologous RNA polymerases I and III has been investigated by gel electrophoresis, hybridization analysis, and RNase T1 mapping. Exogenous RNA polymerase I selectively transcribes rRNA genes. The transcription of these genes by polymerase I is 30- and 8-fold more selective than RNA polymerase III and Escherichia coli polymerase holoenzyme, respectively. Exogenous RNA polymerase III synthesized RNAs similar in size to authentic 5 S RNA, 4.5 S pre-tRNA, and 4 S tRNA. Eleven per cent of this RNA is 5 S RNA as determined by hybridization. Neither polymerase I nor E. coli polymerase synthesizes detectable quantities of RNA in this size range. AT1 ribonuclease digestion of 5 S RNA synthesized by exogenous RNA polymerase III acting on acid-treated chromatin gives a fragment pattern corresponding to that of 5 S RNA. Thus, RNA polymerase III transcribes the entire 5 S gene in this system.