Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of σ ⁷⁰ factors of bacterial RNA polymerases in Arabidopsis thaliana

Abstract

Genes for -like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they probably play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs, designated SIG1, SIG2, and SIG3, for polypeptides possessing amino acid sequences for domains conserved in 70 factors of bacterial RNA polymerases from the higher plant Arabidopsis thaliana. Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green f luorescent protein demonstrated that N-terminal regions of the SIG2 and SIG3 ORFs could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and were induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of our previous analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, these results indicate that expressed levels of the genes may inf luence transcription by regulating RNA polymerase activity in a green tissue-specific manner.The chloroplast is a semi-autonomous organelle whose genetic information is encoded in the nuclear and plastid genomes. The plastid genome encodes genes for photosynthesis, as well as genes for housekeeping functions such as protein synthesis. There is evidence that photosynthesis genes are transcribed by a multimeric Escherichia coli-type RNA polymerase (RNAP), and that housekeeping genes are transcribed by a monomeric T7 or T3 bacteriophage-type RNAP (1). The Ϫ10 and Ϫ35 sequences, 5Ј-TATAAT-3Ј and 5Ј-TTGACA-3Ј, respectively, in promoters of many plastid genes (2) are recognized by the E. coli-type RNAP (3). The E. coli RNAP is composed of a core complex of ␣, ␤, and ␤Ј subunits and one of a variety of factors, the principal one being 70, which is capable of binding to the Ϫ10 and Ϫ35 sequences (4-6). Determination of the complete nucleotide sequences of plastid genomes from liverwort (7), tobacco (8), rice (9), and other plants has resulted in finding of genes, rpoA, rpoB, and rpoC, probably encoding ␣, ␤, and ␤Ј subunits, respectively, of a plastid RNAP. In higher plants, rpoC is duplicated, rpoC1 for ␤Ј subunit and rpoC2 for ␤Љ subunit. Amino acid sequences deduced from maize plastid genes, rpoC2, rpoB, rpoC1, and rpoA, have proved to correspond to those determined chemically of 180-, 120-, 78-, and 38-kDa polypeptides, respectively, of highly purified maize plastid RNAP (10,11).No homolog for the bacterial-like factor of RNAP has been detected in the plastid genomes so far sequenced. However, some reports indicate that plastid -like factors (SLFs) exist in higher plants. Antibodies against an E. coli 70 homolog from the cyanobacterium Anabaena sp. PCC7120 have cross-reacted with polypeptides in purified plastid RNAPs from maize and rice (12). In spinach 90-and 33-kDa polypeptides have been identified in a plastid RNAP that a...

Evolutionary Conservation of C-terminal Domains of Primary Sigma70-type Transcription Factors between Plants and Bacteria

“…Three different cDNAs coding for potential plastid-localized sigma 70 -type transcription initiation factors have recently been cloned and sequenced from A. thaliana (9,23), and the existence of a multigene family of sigma factors has been suggested for Zea mays (24). Meanwhile, the list of Arabidopsis sigma -FIG.…”

“…8) are not found on the plastid genomes (7,1). These genes have been transferred to the nucleus during evolution (9).…”

“…Only recently, six different cDNAs showing strong sequence similarity with genes coding for prokaryotic-like sigma 70 -type initiation factors have been cloned and sequenced from Arabidopsis thaliana (accession numbers: SIG1, dbj: AB004820 and emb: Y15362; SIG2, dbj: AB004821 and emb: Y14567; SIG3, dbj: AB004822; SIG4, gb: AF101075; SIG5, emb: Y18550; and SIG6, emb: AJ250812). For three of them, it has been shown that the corresponding proteins are transported into the chloroplasts (9,14), but the function of these putative transcription factors in the regulation of plastid gene expression is not clear. In particular, it is not clear why several sigma 70 -type factors have been conserved during evolution, despite an important reduction of (plastid) genome size.…”

AtSig5 Is an Essential Nucleus-Encoded Arabidopsis σ-Like Factor

“…Therefore, it is assumed (although not yet experimentally demonstrated) that these nucleus-encoded plant -factors assemble in plastids with the PEP RNA polymerase to effect promoter recognition and transcription initiation. Consistent with this model, many plant -factors contain transit peptides capable of targeting the proteins into chloroplasts (Isono et al, 1997;Kanamaru et al, 1999;Lahiri et al, 1999;Fujiwara et al, 2000;Lahiri and Allison, 2000). Interestingly, the maize -factor, ZmSig2B, accumulates not only in chloroplasts but also in mitochondria, and contains an NH 2 -terminal sequence able to target fused green fluorescent protein (GFP) into mitochondria in transient expression assays (Beardslee et al, 2002).…”

Molecular signatures in protein sequences that are characteristic of cyanobacteria and plastid homologues

“…E). A divergent form of s 70 -like protein is found in plastids (Isono et al, 1997); however, the insert is not present in such homologues. The last of the cyanobacterial signatures described here is a 3 aa insert in the enzyme FtsH protease, involved in the degradation of membrane proteins (Akiyama, 2002) ( Supplementary Fig.…”