The 3′ untranslated regions of chloroplast genes inChlamydomonas reinhardtii do not serve as efficient transcriptional terminators

Abstract

A general characteristic of the 3' untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3' end processing signals in spinach chloroplasts, and in the atpB mRNA of Chlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3' ends of chloroplast genes in Chlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments using Chlamydomonas chloroplast transformants in which different 3' ends were inserted into the chloroplast genome between a petD promoter and a reporter gene. The results showed that none of the 3' ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3' ends of mature mRNAs in Chlamydomonas chloroplasts are formed by 3' end processing of longer precursors.

Blocking polyadenylation of mRNA in the chloroplast inhibits its degradation

“…RNAs that terminate with a stem-loop structure were poorly polyadenylated in vitro, and RT-PCR clones of poly(A)-rich sequences at the end of the mRNA molecule (characterized by a stem-loop structure) were obtained much less frequently than those having the addition site inside the coding region (Lisitsky et al, 1996). Therefore, we suggest that the stem-loop structures characterizing most of the chloroplast mRNAs and shown to be effective 3' end processing signals (Blowers et aL, 1993;Rott et aL, 1996;Stern and Gruissem, 1987;Stern and Kindle, 1993;Stern et al, 1991) also serve as poor polyadenylation sites in order to prevent exonucleolytic degradation (Lisitsky et aL, 1996). Following the endonucleolytic cleavage(s), the proximal fragments are polyadenylated by the addition of (b) The initial step in the mRNA degradation process is suggested to be an endonucleolytic cleavage(s) by as yet unknown endonuclease(s).…”

In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

“…DAAD and DG2 are chimeric genes in which the petD promoter and 5Ј UTR have been fused to aadA (encoding aminoglycoside adenine transferase) and uidA (encoding β-glucuronidase), respectively (Rott et al, 1996;Sakamoto et al, 1993). These genes were introduced into the chloroplast genomes of the mutant strains by biolistic cotransformation with an rDNA antibiotic resistance marker for selection.…”

“…The left part of the gel shows protection of a 210 nt fragment representing the 3Ј end of the petD transcript only in the wild-type strains (wt and 33), while a 73 nt tRNA Arg fragment was protected in all three strains. The mcd1-1 mutation does not affect tRNA Arg accumulation, probably because 5Ј tRNA processing is rapid and therefore precedes 5Ј→3Ј exonucleolytic degradation, and also because transcription of the trnR gene is likely driven by a tRNA-specific promoter (Rott et al, 1996). Fragments protected by the 5Ј polyG-containing probe are predicted to be 90 nt and 130 nt in length for 5Ј ends at the polyG sequence, and the ϩ1 position of the petD 5Ј UTR, respectively.…”

“…Construction of the plasmids pDG2 and pDAAD has been described previously (Rott et al, 1996;Sakamoto et al, 1993). These plasmids contain reporter gene cassettes in which the petD 5Ј UTR is fused to the coding regions of the E. coli genes uidA and aadA, respectively.…”

“…However, there are several important ways in which chloroplast and prokaryote gene expression mechanisms differ. For example, most evidence suggests that the inverted repeat sequences that occur at the 3Ј ends of many chloroplast transcripts do not function in transcription termination as they do in prokaryotes, but rather participate in 3Ј end formation and stabilization (Rott et al, 1996;Stern and Gruissem, 1987;Stern and Gruissem, 1989;Stern and Kindle, 1993). Many mRNAs do not contain canonical Shine-Dalgarno sequences (Sakamoto et al, 1994a; reviewed in Stern et al, 1997), suggesting differences in translation initiation mechanisms.…”