Nascent Transcript Cleavage Research Articles

Mechanism of DmS-II-mediated pause suppression by Drosophila RNA polymerase II.

Transcription elongation factor S-II mediates nascent transcript cleavage by RNA polymerase II (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). We have examined the mechanism of action of the Drosophila S-II analog, DmS-II, in a defined transcription system. Our results show that DmS-II is necessary and sufficient to activate nascent transcript cleavage by RNA polymerase II during transcription of a dC-tailed template. The pattern of transcripts resulting from prolonged action by DmS-II indicates that there are kinetic barriers to transcript shortening. During the cleavage reaction, the polymerase remains in register with the template strand and generates mainly nucleotide dimers. The ability of DmS-II to mediate transcript shortening resides in the carboxyl-terminal half of the protein. Our results support a model for pause suppression in which DmS-II binds to the paused polymerase, causes one cleavage event and is then released from the complex. Elongation by the polymerase then allows a second encounter with the pause site and a second chance of passing the site. Complete pause suppression may require multiple transcript shortening events for some polymerase molecules.

Structure of a new nucleic-acid-binding motif in eukaryotic transcriptional elongation factor TFIIS.

Transcriptional elongation involves dynamic interactions among RNA polymerase and single-stranded and double-stranded nucleic acids in the ternary complex. In prokaryotes its regulation provides an important mechanism of genetic control. Analogous eukaryotic mechanisms are not well understood, but may control expression of proto-oncogenes and viruses, including the human immunodeficiency virus HIV-1 (ref. 8). The highly conserved eukaryotic transcriptional elongation factor TFIIS enables RNA polymerase II (RNAPII) to read though pause or termination sites, nucleosomes and sequence-specific DNA-binding proteins. Two distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate read-through and possibly nascent transcript cleavage. Here we describe the three-dimensional NMR structure of a Cys4 nucleic-acid-binding domain from human TFIIS. Unlike previously characterized zinc modules, which contain an alpha-helix, this structure consists of a three-stranded beta-sheet. Analogous Cys4 structural motifs may occur in other proteins involved in DNA or RNA transactions, including RNAPII itself. This new structure, designated the Zn ribbon, extends the repertoire of Zn-mediated peptide architectures and highlights the growing recognition of the beta-sheet as a motif of nucleic-acid recognition.

Elongation factor SII-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein.

In eukaryotes the genetic material is contained within a coiled, protein-coated structure known as chromatin. RNA polymerases must recognize specific nucleoprotein assemblies and maintain contact with the underlying DNA duplex for many thousands of base pairs. Template-bound lac operon repressor from Escherichia coli arrests RNA polymerase II in vitro and in vivo [Kuhn, A., Bartsch, I. & Grummt, I. (1990) Nature (London) 344, 559-562; Deuschele, U., Hipskind, R. A. & Bujard, H. (1990) Science 248, 480-483]. We show that in a reconstituted transcription system, elongation factor SII enables RNA polymerase II to proceed through this blockage at high efficiency. lac repressor-arrested elongation complexes display an SII-activated transcript cleavage reaction, an activity associated with transcriptional read-through of a previously characterized region of bent DNA. This demonstrates factor-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein. Nascent transcript cleavage may be a general mechanism by which RNA polymerase II can bypass many transcriptional impediments.

The RNA polymerase II elongation complex. Factor-dependent transcription elongation involves nascent RNA cleavage.

Regulation of transcription elongation is an important mechanism in controlling eukaryotic gene expression. SII is an RNA polymerase II-binding protein that stimulates transcription elongation and also activates nascent transcript cleavage by RNA polymerase II in elongation complexes in vitro (Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). Here we show that SII-dependent in vitro transcription through an arrest site in a human gene is preceded by nascent transcript cleavage. RNA cleavage appeared to be an obligatory step in the SII activation process. Recombinant SII activated cleavage while a truncated derivative lacking polymerase binding activity did not. Cleavage was not restricted to an elongation complex arrested at this particular site, showing that nascent RNA hydrolysis is a general property of RNA polymerase II elongation complexes. These data support a model whereby SII stimulates elongation via a ribonuclease activity of the elongation complex.

Rapid initial cleavage of nascent pre-rRNA transcripts in yeast

In yeast cells, as in many other eukaryotes, the initial step in the processing of the pre-rRNA primary transcript is removal of external transcribed spacer (ETS) sequences from the 5′ end of the transcript. We show here, both by Northern analysis and by quantitative hybridization procedures using cloned yeast ETS sequences, that in cells growing exponentially at 23 °C most nascent pre-rRNA transcripts no longer contain ETS sequences. Moreover, quantitative hybridization shows that uncleaved pre-rRNA molecules that still contain ETS sequences have a half-life of only 0.5 minute, a value that supports the finding that ETS removal usually takes place before pre-rRNA transcription is complete. Under these same conditions, the half-life of ETS sequences is shown to be only 1.0 minute.

Some ultrastructural aspects of genetic activity in eukaryotes.

A brief overview is given of the types of information that have been obtained by chromatin spreading methods regarding the ultrastructure and regulation of genetic units. The examples used are: ribosomal RNA genes of the spotted newt; the silk fibroin gene of Bombyx mori; chorion gene amplification in Drosophila melanogaster; RNA synthesis patterns during early embryogenesis of D. melanogaster; regulation of transcription on homologous non-ribosomal transcription units of D. melanogaster; specific nascent transcript cleavage in insects; and nascent polypeptides on insect polyribosomes. The conclusion is drawn that further innovations will be required before significant advances in the use of chromatin spreading techniques to study ultrastructural aspects of genetic activity can occur.

Correlation of hnRNP structure and nascent transcript cleavage

Using electron microscopy of spread chromatin, we have observed nonnucleolar transcription units from Drosophila melanogaster and Calliphora erythrocephala that display specific cleavage of nascent transcripts. We have quantitatively analyzed 20 of these relatively long transcription units. The primary RNP structure of homologous transcripts is nonrandom with respect to both RNA sequence and the cleavage event. In general, released RNA fragments have a smooth fibrillar RNP morphology (∼50 Å wide) and retained segments have a thicker particulate morphology (∼250 Å diameter). A characteristic secondary structure formation also accompanies cleavage—that is, RNP fibril loops form by association of noncontiguous transcript sequences that correspond to the terminal regions of the segment to be released. RNP particles form at the loop base sequences prior to their association and apparently coalesce upon loop formation. These loops, and thus the released segments, range in length between 1 and 25 kb on the examples we have analyzed. Cleavage of nascent hnRNA transcripts appears to be a fairly common event in these organisms and occurs within 0.3–3 min after transcription of the cleavage site.