Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two specific sites.

نویسندگان

  • A Fels
  • K Hu
  • D Riesner
چکیده

Pospiviroidae, with their main representative potato spindle tuber viroid (PSTVd), are replicated via a rolling circle mechanism by the host-encoded DNA-dependent RNA polymerase II (pol II). In the first step, the (+)-strand circular viroid is transcribed into a (-)-strand oligomer intermediate. As yet it is not known whether transcription is initiated by promotors at specific start sites or is distributed non-specifically over the whole circle. An in vitro transcription extract was prepared from a non-infected potato cell culture which exhibited transcriptional activity using added circular PSTVd (+)-strand RNA as template. In accordance with pol II activity, transcription could be inhibited by alpha-amanitin. RT-PCR revealed the existence of at least two different start sites and primer extension identified these as nucleotides A(111) and A(325). The sequences of the first 7 nt transcribed are very similar, (105)GGAGCGA(111) and (319)GGGGCGA(325). GC-boxes are located at a distance of 15 and 16 nt upstream, respectively, in the native viroid structure, which may act to facilitate initiation. The GC-boxes may have a similar function to the GC-rich hairpin II in the (-)-strand intermediate, as described previously. The results are compared with the corresponding features of avocado sunblotch viroid, which belongs to a different family of viroids and exhibits different transcription initiation properties.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Synthesis of (+) and (-) RNA molecules of potato spindle tuber viroid (PSTV) in isolated nuclei and its impairment by transcription inhibitors.

Transcription studies with highly purified potato cell nuclei in combination with a 'transcription-hybridization analysis' unequivocally demonstrate that the nucleus is the subcellular site where the entire process of PSTV replication takes place. Inhibition experiments with actinomycin D and alpha-amanitin furthermore suggest that the nuclear DNA-dependent RNA polymerases I and II are involved...

متن کامل

Detection and analysis of hairpin II, an essential metastable structural element in viroid replication intermediates.

In (-)-stranded replication intermediates of the potato spindle tuber viroid (PSTVd) a thermodynamically metastable structure containing a specific hairpin structure (HP II) has been proposed to be essential for viroid replication. In the present work a method was devised allowing the direct detection of the HP II structure in vitro and in vivo using a biophysical approach. An RNA oligonucleoti...

متن کامل

Accumulation of Potato spindle tuber viroid-specific small RNAs is accompanied by specific changes in gene expression in two tomato cultivars.

To better understand the biogenesis of viroid-specific small RNAs and their possible role in disease induction, we have examined the accumulation of these small RNAs in potato spindle tuber viroid (PSTVd)-infected tomato plants. Large-scale sequence analysis of viroid-specific small RNAs revealed active production from the upper portion of the pathogenicity and central domains, two regions prev...

متن کامل

Comparative studies of two viroids: analysis of potato spindle tuber and citrus exocortis viroids by RNA fingerprinting and polyacrylamide-gel electrophoresis.

Highly purified isolates of potato spindle tuber viroid (PSTV) from Lycopersicon esculentum cv. Rutgers and citrus exocortis viroid (CEV) from Gynura aurantiaca have been compared. Two-dimensional fingerprinting analysis of PSTVand CEV-RNA labeled in vitro with lzrI has demonstrated that (i) each of these RNA species has a complexity compatible with the size estimate of 250X350 nucleotides and ...

متن کامل

Replicating potato spindle tuber viroid RNA is accompanied by short RNA fragments that are characteristic of post-transcriptional gene silencing.

The low molecular weight fraction of tomato plants inoculated with potato spindle tuber viroid (PSTVd) contains a population of short PSTVd-specific RNAs of either polarity. The main constituents were RNAs of 22 and 23 nt representing different domains of the viroid genome. The occurrence of such distinct RNA species indicated that the nuclear replicating PSTVd RNA induces post-transcriptional ...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • Nucleic acids research

دوره 29 22  شماره 

صفحات  -

تاریخ انتشار 2001