Site of premature termination of late transcription of simian virus 40 DNA: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

156

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

mentioning

confidence: 99%

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

Henikoff¹,

Cohen²

1984

156

“…Above the line map are the results for a typical transcription of normal nuclei, and below the map are the results for nuclei from DRB-treated cells. Treatment of cells with DRB commonly induces pausing or premature termination, thus augmenting promoter-proximal transcription in relation to promoter-distal transcription (35,41,51,52). Both the pH_ and PA-globin genes exhibit this response such that transcription past exon 2 is diminished in nuclei isolated from DRBtreated cells, but promoter-proximal transcription is augmented (data not shown).…”

Section: Resultsmentioning

confidence: 94%

Transcription termination at the chicken beta H-globin gene.

Pribýl

Martinson

1988

We characterized the transcription termination region of the chicken 3H.-globin gene. First we located the region by nuclear runon transcription in vitro. Then we sequenced and subcloned it into a chloramphenicol acetyltransferase (CAT) expression vector for assay in vivo. The region of PH termination contains two interesting elements located about 1 kilobase downstream of the pH gene poly(A) site. Either element alone can block CAT expression if inserted between the promoter and the poly(A) site of the cat gene in pRSVcat. The first element in the termination region is an unusually large inverted repeat in the DNA (AG = -71 kcal). The second element, 200 base pairs further downstream, is an RNA polymerase II promoter which directs transcription back upstream on the complementary strand. This transcription converges on and collides with that from the 13H gene at or near the inverted repeat where transcription from both directions stops. We propose that the inverted repeat is a strong pause site which positions the converging polymerases for mutual site-specific termination.Transcription termination signals have been identified and studied extensively both in vivo and in vitro for Escherichia coli RNA polymerase and eucaryotic RNA polymerases I and III (4, 26-28, 37, 44). In E. coli, two main types of termination are recognized, factor dependent and factor independent (44). Factor-dependent termination signals precede the actual sites of termination and exhibit considerable sequence flexibility, which makes them difficult to recognize. A protein factor, usually p, interacts with the RNA to assist termination by the polymerase. Factor-independent terminators, in contrast, have well-defined sequence signals and are readily recognizable by their G+C-rich palindrome, which precedes a cluster of dT residues in the nontemplate strand of the DNA at the site of termination. Additional modes of procaryotic transcription termination also occur and may be of physiological significance. These include transcriptional interference of converging (33) or tandem (1) transcription units and transcriptional arrest caused by blockage of the DNA template by a bound factor (50).In eucaryotes, most RNA polymerase III terminators, like the factor-independent terminators in E. coli, possess a dT cluster and exhibit no obligatory requirement for a factor, but they lack a palindrome (37, 44). Eucaryotic RNA polymerase I termination (27), although factor dependent, bears little resemblance to p-dependent termination in E. coli but instead resembles aspects of the protein blockage mechanism described by Sellitti et al. (50). For polymerase I terminators, it is the DNA to which the factor binds (27), and the signal follows, rather than precedes, the site of termination (27,28).RNA polymerase II appears to incorporate elements of all the above termination mechanisms in its repertoire of strategies. Transcription of the human gastrin gene appears to terminate upstream of the termination signal as for RNA polymerase I (3, 49). The terminator for...

“…2 and 7). These results suggest that interactions similar to those involved in procaryotic transcription termination (1,3,4,10,13,16) reports (6,15) suggest that a DNA sequence in simian virus 40 resembling a procaryotic transcription terminator is an in vivo transcription termination site for eucaryotic RNA polymerase II (Fig. 8).…”

mentioning

confidence: 84%

“…In higher eucaryotes, however, it appears that polyadenylation and termination are not coupled, and in fact, with few exceptions, it is not known where termination of these transcripts occurs. The in vivo 3' termini of nuclear RNA polymerase lI transcripts have been accurately determined for a number of histone genes (7), human Ul RNA (12), and the simian virus 40 major late transcript (6,15). In these cases, transcription appears to terminate after a region of dyad symmetry in the DNA template.…”

mentioning

confidence: 99%

Pausing and termination of human RNA polymerase II transcription at a procaryotic terminator.

Hatfield¹,

Sharp²,

Rosenberg³

1983

Kinetic analyses of runoff transcription in a cell-free eucaryotic transcription system revealed that the bacteriophage lambda 4S RNA terminator caused human RNA polymerase II to pause on the template and partially terminate transcription of transcripts initiated by the adenovirus 2 major late promoter. Analogous to the procaryotic RNA polymerase, the eucaryotic enzyme terminated just beyond the guanine-plus-cytosine-rich region of dyad symmetry in the terminator sequence. These results suggest that the eucaryotic RNA polymerase II may respond to transcription termination sequences similar to those used by the procaryotic enzyme. However, similar templates containing lambda tint or lambda tR. terminators did not elicit pausing or termination, suggesting that other features, such as sequence specificity, may also be involved.In eucaryotes the 3' termini of most RNA polymerase II transcripts are generated by RNA processing and subsequent polyadenylation (5). In yeasts, the polyadenylation of the iso-1-cytochrome c transcript is coupled to the termination event (17). In higher eucaryotes, however, it appears that polyadenylation and termination are not coupled, and in fact, with few exceptions, it is not known where termination of these transcripts occurs. The in vivo 3' termini of nuclear RNA polymerase lI transcripts have been accurately determined for a number of histone genes (7), human Ul RNA (12), and the simian virus 40 major late transcript (6, 15). In these cases, transcription appears to terminate after a region of dyad symmetry in the DNA template. This same type of DNA structure is found in procaryotic termination sequences and has been well defined (13).Termination sites at the end of procaryotic genes are specified by a DNA sequence, including a guanine-plus-cytosine (G+C)-rich region of dyad symmetry, followed by a string of thymidine residues. Termination of transcription occurs within the string of thymidine residues, resulting in an RNA transcript with a G+C-rich stem loop structure, followed by a short string of uridine residues at the 3' terminus of the RNA transcript. A wide variety of experimental results support the hypothesis that the G+C sequences in the DNA impede the progress of the procaryotic RNA polymerase; the stem loop formation in the transcript triggers the termination event, and the instability of the deoxyadenosine-uridine base pairing between the DNA template and the RNA transcript facilitates the release of the transcript. Thus, it appears that the mechanism of transcription termination in procaryotes is based upon the physical chemistry of nucleic acid interactions specified by the DNA sequence and the secondary structural properties of the RNA transcript (1, 3, 4, 10, 16).It is not unreasonable to assume that these same physicochemical parameters might influence the termination of RNA polymerase II transcription in eucaryotes. Therefore, we investigated whether procaryotic termination sites also function as eucaryotic transcription terminators. We found that the procaryotic p...