Sequences responsible for transcription termination on a gene segment in Saccharomyces cerevisiae.

Abstract

We have mapped a signal sequence for mRNA 3'-end formation in Saccharomyces cerevisiae by using a Drosophila melanogaster DNA segment that complements a yeast adenine-8 mutation. That the 3' end of the transcript in S. cerevisiae nearly coincides with that in D. melanogaster is consistent with the possibility that mRNA termini are similarly determined in both organisms. Deletion analysis reveals that the complete signal is no more than 21 base pairs long. Part of the signal is the sequence TTTTTATA, which is seen in the termination region of several yeast genes. TTTTTATA appears to be able to act autonomously as a partial termination signal. The efficiency of the complete signal is affected by substitution of sequences downstream from it. This modulation of the effect of a signal is consistent with termination in S. cerevisiae, resembling rhodependent termination in bacteria.Until recently, transcription termination in eucaryotes has been poorly understood, in part because of the rapid processing that occurs at the 3' ends of transcripts for proteincoding genes. Processing usually involves cleavage of a precursor followed by polyadenylation in higher eucaryotes (6, 21). Histone mRNA 3' ends which lack polyadenylic acid were once thought to result from transcription termination (3) but now appear to be generated by processing (2, 7). The biochemical elucidation of mRNA 3' processing should be possible with the recent development of a cell-free system (23). In contrast to processing, actual transcription termination, which can occur at a considerable distance downstream from the mRNA 3' end (6, 27), has been relatively refractory to analysis.In Saccharomyces cereisisiae, polyadenylic acid addition and transcription termination appear to be coupled, as essentially all transcripts from protein-coding genes are polyadenylated [poly(A)+] (29). Therefore, the strong possibility exists that the study of 3'-end formation of an mRNA in yeast cells will shed light on the actual transcription termination event. Termination is known to be important in the regulation of a variety of bacterial operons (reviewed in references 17 and 24) and appears to be involved in the regulation of at least one eucaryotic gene (9, 10, 28). We have been investigating the sequence determinants of transcription termination on a DNA segment in S. cerev,isiae. This segment is derived from Drosophila melanogaster and complements a yeast adenine-8 mutation (15). We showed previously that mRNA 3' ends actually occur 50 to 90 base pairs (bp) downstream from a control signal, the 3' boundary of which we mapped precisely (13). Here we show that this signal is no more than 21 bp long. We also present evidence suggesting that an 8-bp portion of this signal promotes partial termination. It

3′-end-forming signals of yeast mRNA

“…TATGTA, and TACA TA-were identified by examining intragenic revertants and oligonucleotide-directed alterations that reverted the cyc1-512 defect (32). Further support for the involvement of these and related sequences in 3Ј end formation came from studies of other altered yeast genes (1,20,21,41) and from the insertion of heterologous fragments into the yeast genome (16,17,22).Further analysis of the deletions of the CYC1 ϩ 38-bp region and the introduction of specific alterations in the cyc1-512 mutant led Russo et al (31) to suggest that 3Ј-end-forming signals are composed of three distinct but interacting elements. The first and second were previously denoted, respectively, as the upstream and downstream elements but are now referred to as, respectively, the efficiency and positioning elements.…”

“…TATGTA, and TACA TA-were identified by examining intragenic revertants and oligonucleotide-directed alterations that reverted the cyc1-512 defect (32). Further support for the involvement of these and related sequences in 3Ј end formation came from studies of other altered yeast genes (1,20,21,41) and from the insertion of heterologous fragments into the yeast genome (16,17,22).…”

Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome.

“…The HindIll site used to construct the GALI-MRP7 gene fusion (Fig. 3 and 8) (18) and that the 3' mRNA sequences fit the consensus CAAGT/CTTTG (19). Although the TTTTTATA consensus was not found anywhere near the 3' end of MRP7, the sequence CAAGCTTTG was found at the end of the open reading frame and included part of the stop codon (underlined in Fig.…”

A specific DNA sequence controls termination of transcription in the gastrin gene.

“…polyadenylation of mRNA. For some yeast genes, when the terminal mRNA sequence is CAATGCTITG, the alternative motif 'I'ITT'ATA has been proposed as a yeast terminator (20,21).…”