Termination Signal For RNA Polymerase Research Articles

NMR evidence for the RNA stem-loop structure involved in the transcription attenuation of E. coli trp operon.

High field 1H-NMR studies of a synthetic 21-mer RNA fragment, corresponding to residues +114 to +134 within the trp leader mRNA transcript, have been carried out. Seven well resolved imino proton resonances corresponding to six C-G and one A-U hydrogen bonded base pairs, together with their characteristic NOE patterns can be identified in the NMR spectrum. This experimental result provides direct evidence for the postulated stem-loop secondary structure, 3:4, which has been reported to act as a transcription termination signal for RNA polymerase.

Delineation of the transcriptional boundaries of the lux operon of Vibrio harveyi demonstrates the presence of two new lux genes.

The 5' and 3' ends of the lux mRNA of Vibrio harveyi, which extends over 8 kilobases, have been mapped, and two new genes, luxG and luxH, were identified at the 3' end of the lux operon. Both S1 nuclease and primer extension mapping demonstrated that the start site for the lux mRNA was 26 bases before the initiation codon of the first gene, luxC. The promoter region contained a typical -10 but not a recognizable -35 consensus sequence. By using S1 nuclease mapping the mRNA was found to be induced in a cell density- and arginine-dependent manner. The DNA downstream of the five known V. harveyi lux genes, luxCDABE, was sequenced and found to contain coding regions for two new genes, designated luxG and luxH, followed by a classical rho-independent termination signal for RNA polymerase. luxG codes for a protein of 233 amino acids with a molecular weight of 26,108, and luxH codes for a protein of 230 amino acids with a molecular weight of 25,326. The termination signal is active in vivo as demonstrated by 3' S1 nuclease mapping, confirming that the two genes are part of the V. harveyi lux operon. Comparison of the luxG amino acid sequence with coding regions immediately downstream from luxE in other luminescent bacteria has demonstrated that this gene may be a common component of the luminescent systems in different marine bacteria.

An undecamer DNA sequence directs termination of human ribosomal gene transcription

Previously we have shown that a repetitive 18 bp sequence motif, the Sal box (AGGTCGACCAGA/TT/ANTCCG), present in the 3' terminal spacer of mouse rDNA constitutes a termination signal for RNA polymerase I (pol I). Similar sequence elements which are functionally analogous to the murine terminator are present in the spacer of human rDNA. However, the human termination signal is shorter encompassing only 11 bp (GGGTCGACCAG) which correspond to the proximal part of the mouse sequence. Two out of the five human Sal box elements are functionally inactive due to natural point mutations which damage factor binding. A similar sequence motif with a 10 of 11 base identity with the downstream terminators is located upstream of the human transcription initiation site. The upstream element interacts with the same factor(s) as the downstream terminators and is also capable to stop elongating human RNA polymerase I. Despite the human and mouse factors exert different electrophoretic mobilities in gel retardation assays, UV-crosslinking and proteolytic clipping experiments indicate that both the sizes and the tertiary structure of the Sal box binding proteins of both species are very similar. When bound to DNA, both the human and the mouse factor terminate transcription of pol I from the heterologous species. The results implicate that changes in signal sequences necessary for termination have been accompanied by compensatory changes in the DNA binding domain of the protein(s) interacting with the termination signal. In contrast, the protein-protein interactions between the termination factor and the transcribing RNA polymerase I appear to have been conserved during evolution.

Characterization of a New Termination Signal for RNA Polymerase III Responsible for Generation of a Discrete-sized RNA Transcribed from Salmon Total Genomic DNA in a HeLa Cell Extract

A new type of termination signal for RNA polymerase III transcription has been identified. A cloned DNA from salmon highly repetitive sequences, designated as Sm2, serves as a template for an RNA with about 140 nucleotides, but the clone does not have four or more consecutive T residues in the presumed termination site. S1 nuclease mapping analysis clearly demonstrated that termination occurred at the beginning of the AT-rich sequence in the 3' region of Sm2. Studies with a series of 3'-deletion mutants strongly suggested that an AT sequence of more than nine nucleotides functioned as a terminator. Furthermore, results with another series of 3'-deletion mutants showed that a sequence of only nine nucleotides (ATATATATT) in the noncoding strand in fact functioned as a terminator (designated as the 9AT terminator), although with relatively low efficiency compared to a tract of TTTT introduced downstream, and that new cryptic termination sites were introduced in the upstream slight AT-rich region by approach of the 9AT terminator. An AT-rich region downstream from the actual termination site of Sm2 DNA may enhance the efficiency of termination by facilitating detachment of RNA polymerase from the template DNA. Since the nucleotide sequence around the termination site appears to be conserved in the salmon repetitive sequences, this new terminator may be responsible for the generation of a discrete-sized RNA transcribed from salmon total genomic DNA in vitro.

Identification of intrinsic termination sites in vitro for RNA polymerase II within eukaryotic gene sequences

We have identified and mapped several DNA sequences within a human histone gene (H3.3) at which in-vitro transcription by highly purified RNA polymerase II is efficiently terminated. Since transcription in our system involves only RNA polymerase II acting on a linear DNA template, these sequences contain “intrinsic” termination signals recognized by the polymerase protein itself. The existence of such signals within a gene suggests that efficient antitermination systems probably exist for mammalian transcription units. Alternatively, there could be a high frequency of premature transcription termination, or “polarity” for genes such as H3.3. Intrinsic transcription termination sites in H3.3 are located in sequences of consecutive thymidylate residues (5 to 8 nucleotides) on the non-transcribed DNA strand (T-runs), from which it is likely that such T-runs are elements of the intrinsic termination signal for RNA polymerase II. However, transcription proceeds without significant termination through many similar T-runs, from which it follows that these intrinsic termination signals include other elements. Since transcription is also terminated efficiently at these sites when the transcript remains bound along its full length as a DNA-RNA hybrid, it is unlikely that formation of specific RNA secondary structures in the transcript is a general element of the intrinsic termination signal. Although DNA sequences downstream from the coding portion of the mouse β-globin gene have been implicated as sites of transcription termination in vivo, these regions do not contain strong intrinsic termination signals, and transcription in vitro proceeds through these regions almost undiminished. Transcriptional termination in this region in vivo may depend on the presence of termination factors or other intracellular elements, and there may be multiple classes of DNA signals that control eukaryotic termination.

Purified lupus antigen La recognizes an oligouridylate stretch common to the 3' termini of RNA polymerase III transcripts.

The small ribonucleoproteins recognized by anti-La sera consist mainly of RNA polymerase III products complexed with an antigenic cellular protein of 50 kd. A biochemical procedure for purifying the La protein from HeLa cells is described. The interaction of the isolated protein with a collection of model tRNA precursors, generated by ligation of specific oligonucleotides to the 3' terminus of yeast tRNAPhe, was studied. The most stable complexes are formed with adducts possessing three or four terminal uridylate residues. Addition of a terminal phosphate, fragmentation of the RNA, or substitution of other nucleotides reduce the affinity for the La protein. The preferred terminal sequence recognized and bound by La protein is homologous to the transcriptional termination signal for RNA polymerase III.