Terminal Stem-loop Structure Research Articles

Poly (A) RNA in Bacillus subtilis: Identification of the polyadenylylation site of flagellin mRNA

Using improved procedures for the synthesis, amplification, and cloning of DNA complementary to bacterial poly(A) RNA, we succeeded in deducing the 3′-terminal sequence of polyadenylylated flagellin mRNA encoded by the Bacillus subtilis hag gene. The site of polyadenylylation was found to be just upstream of the terminal stem-loop structure corresponding to the putative rho-independent transcription terminator of the hag gene. This corresponds to the major polyadenylation site of Escherichia coli lipoprotein (lpp) mRNA [G.C. Gao and N. Sarkar (1992) Proc. Natl. Acad. Sci. USA 89, 7546–7550], suggesting that the primary transcript is processed by endonucleolytic cleavage prior to polyadenylylation in Gram-positive as well as in Gram-negative bacteria.

Specific binding of host cell proteins to the 3'-terminal stem-loop structure of rubella virus negative-strand RNA

At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed.

Studies of local stability in histone, U-snRNA and globin precursor mRNAs around transcription termination sites

Using an efficient algorithm we have carried out extensive studies of the local distributions of significant stem-loop structures around the 3′ termini of histone, U-snRNA and globin precursor mRNAs. We have analyzed statistically about 320 distribution curves from 47 species of histone, 16 species of U-snRNA and 22 globin sequences. Our results show that there are common and significant stem-loop structures near the 3′ ends of histone mRNAs of vertebrates and invertebrates. One of these structures encompasses both of the features known to be essential for 3′ processing: the palindrome and the CAAGAAAGA consensus sequence. Such stem-loop structures are not found in the 3′ termini of yeast and wheat histones, in globin precursor mRNAs or near the 3′ end of U-snRNA of vertebrates. These results are consistent with recent experimental data, in which the terminal stem-loop structure and the CAAGAAAGA spacer within the histone pre-mRNAs are required absolutely for RNA processing. For globin pre-mRNAs a common secondary structure appears to recur approx. 40 bases downstream from the conserved AAUAAA motif. This is also in agreement with experimental data which shows that the sequence downstream of the polyadenylation site is important for 3′ end formation. Our results are also compatible with the suggestion that the 3′ ends of histone and U-snRNA may be generated by related, but distinct, RNA processing mechanisms.

The terminal RNA stem-loop structure and 80 bp of spacer DNA are required for the formation of 3′ termini of sea urchin H2A mRNA

We have studied the exact sequence requirement for the formation of 3′ termini of the sea urchin H2A mRNA in frog oocyte injection experiments. Point mutations destroying the symmetry of the inverted DNA repeat in the mRNA trailer coding sequences prevent the generation of genuine 3′ termini. Mutants containing complementary base changes are pseudorevertants and allow the production of H2A mRNA with faithful 3′ termini at wild-type levels. Our transcription analyses show that it is primarily the sequence of the transcribed strand that decides whether or not true 3′ mRNA termini are produced. This is evidence that an RNA stem-loop structure, rather than a DNA cruciform, is essential for this process. Spacer sequences are absolutely required, because in their absence only H2A mRNA with spacer transcript extensions are found. Once the canonical CAAGAAAGA and flanking sequences are linked to the H2A gene, H2A messenger is synthesized at a suboptimal rate, which can be increased to wild-type levels by the addition of 80 bp of the spacer immediately adjacent to the H2A gene.