The bacteriophage T7 and SP6 RNA polymerases terminate transcription on two types of termination signal without help of any factors. One of the two types of intrinsic termination mechanism involves an RNA hairpin structure and the other does not. Some novel properties of the two mechanisms are characterized here in detail.
Termination efficiency of the bacteriophage T7 RNA polymerase at the intrinsic, hairpin-dependent terminator of bacteriophage T7 can be reduced by the G-rich abortive transcripts produced from the T7 promoter [Kwon, Y.-S. (1993) Master Thesis, KAIST, Taejon, Republic of Korea]. To identify the mechanism of termination efficiency reduction by the G-rich abortive transcripts, the specificity of the termination efficiency reduction was examined. Termination efficiency was reduced only for the T7 terminator. All the other terminators examined show constant termination efficiencies regardless of the presence of such abortive transcripts. When the transcribing RNA polymerases were other phage-encoded RNA polymerases, the abortive transcripts reduced termination efficiency at the T7 terminator. The efficiency reduction by abortive transcripts was abolished when mutations were introduced in a region encoding the base part of hairpin structure in nascent transcripts. However, mutations in other parts do not invoke the resistance to the influence of abortive transcripts on termination efficiency. Thus, the abortive transcripts produced from the T7 promoter appear to interfere with termination through base-specific interaction. The region of nascent transcript that interacts with the abortive transcripts constitutes a part of the essential hairpin-forming secondary structure.
The transcript off the T7 terminator can form a stable hairpin structure, followed by a run of rU. In order to identify the elements that are required for termination, the termination efficiencies of T7 RNA polymerase, SP6 RNA polymerase and nicked SP6 RNA polymerase at a variety of T7 terminator mutants were determined. Termination occurs efficiently only after the synthesis of an RNA product with the stable secondary structure and a run of U residues. The reduced stability of stem-loop structure of RNA transcript reduces the termination efficiency. However, restored structures by introducing compensatory mutations hardly recover the original termination efficiency. Thus, these results clearly show that stable secondary structure is an important determinant in termination-elongation decision but other factors influence the termination efficiency as much as the secondary structure does. A proteolytic nick at the N-terminal domain of SP6 RNA polymerase reduces processivity of elongating ternary complex and increases termination efficiency at all the examined terminators that can form a hairpin structure in nascent transcripts. The results are consistent with the previous proposal that the N-terminal domain of T7 RNA polymerase is important for processivity of elongation complex, and may stabilize elongation complex. Thus, the increase in termination efficiency appears to directly reflect the reduction in stability of elongation complex, and termination process involves destabilization of elongation complex.
A factor-independent terminator T1 in the E. coli operon rrnB can stop elongation of not only E. coli but also bacteriophage RNA polymerases. It shows the typical structural properties of prokaryotic intrinsic terminators. The termination sites at the terminator T1 were determined by comparing the mobilities of the terminated transcripts with sequence-specific RNA ladders made by incorporation of 3-dNTPs during transcription. The termination sites (called T1a) lie at the oligo(U) stretch and the other sites (called T1b) were far away after the run of rU. The sites at which T7 RNA polymerase stops differ from previous report and termination sites for SP6 RNA polymerase are different from those for T7 RNA polymerase. The replacement of GTP with ITP in transcription reaction interferes with termination at oligo(U) stretch and stimulates termination at the downstream site. The termination at the sites that lie in the U stretch depends on stable hairpin structure, and nicked SP6 RNA polymerase stops at the site with increased efficiency. All the properties of termination at oligo(U) are similar to termination at the T7 terminator.
On the contrary, termination at downstream of oligo(U) is independent of secondary structure, and the nicked SP6 polymerase reads through the termination signal. When the hairpin RNA-forming region was deleted from the terminator T1, it still efficiently terminates the SP6 RNA polymerase, and the termination site on the deletion templates is the same as that on the original terminator. The minimal template sequence that can induce transcription termination of the SP6 RNA polymerase is CGTTTTATCTGTTGTTTG. In this study, at least three elements are identified essential for termination of transcription by the SP6 RNA polymerase. The conserved ATCTGTT sequence in performs pivotal roles in termination site selection. All mutations in the conserved sequence abolish termination. Non-template stand sequence immediately upstream of the conserved region is important for efficient termination. Some sequence variations in non-template strand abolish or weaken termination. The sequence of template strand is not as important as that of non-template strand, although the presence of complementary template strand is necessary for efficient termination. The presence of uridine-rich stretch in transcript RNA located in the immediately upstream region of the termination site is essential for termination. Thus, instability of base pairing between rU-dA is required for termination. The oligo(U) stretch needs to be interrupted by other bases in order to suppress transcription slippage. The termination site of active terminator variants is always same distance downstream from the conserved sequence. On the other hand, transcription by the T7 RNA polymerase is not terminated efficiently on the terminator variants that do not encode a hairpin structure, and termination sites differ from the terminator that forms a hairpin structure. The results suggest that termination of T7 enzyme at T1b site requires upstream elements, although it dose not require stable RNA secondary structure in nascent transcript.
박테리오 파아지 T7과 SP6의 RNA 중합효소는 다른 단백질 인자의 도움 없이도 두 가지 형태의 종결신호에 의해 전사가 종결된다. 본 연구를 수행하여 두 가지 종결기작의 특성을 규명하였다.
박테리오 파아지 T7의 전사종결신호는 T7 전사 촉진제로부터 만들어지는 구아닌이 많은 전사개시 중단물에 의해 종결효율이 감소한다. 종결효율 감소의 기작을 규명하기 위해 전사 개시 중단물에 의한 종결효율 감소는 T7 종결 신호에서만 관찰되었다. 다른 종결신호는 전사개시 중단물에 의한 영향을 받지 않았다. T7 전사종결신호에 돌연변이를 도입하면 일부 돌연변이 종결신호는 전사개시 중단물 RNA의 영향을 받지 않게 된다. 이 결과는 전사개시 중단물과 전사물 사이의 특이적 상호작용에 의해서 종결효율 감소가 이루어짐을 암시한다. 파아지 T7의 전사종결신호는 안정한 머리핀 구조를 형성할 수 있으며 여러 개의 우라실 잔기를 가지고 있다. 전사종결에 필수적인 요소들을 규명하기 위하여 돌연변이 종결신호에서 T7 RNA 중합효소의 종결효율을 측정하였다. 효율적인 전사종결은 안정된 머리핀 구조를 필요로 하지만 모든 안정된 구조가 효율적인 전사종결을 유발하는 것은 아니다. 이 결과는 전사물과 RNA 중합효소 사이의 상호작용이 전사종결에서 중요함을 강하게 시사한다.
대장균의 rrnB 오페론의 T1 종결신호는 머리핀 구조를 필요로 하는 T1a라는 종결신호와 머리핀 구조를 필요로 하지 않는 T1b 종결신호를 가지고 있다. 본 연구의 결과는 T1a는 머리핀 구조를 필요로 하는 종결신호의 전형적인 특성을 모두 지니고 있음을 보여준다. 반면에 T1b 종결신호는 완전히 다른 기작으로 전사종결이 이루어진다. 머리핀 구조를 제거한 주형에서도 T1b 종결신호는 작동하며 N-terminal domain이 변형된 중합효소는 이 신호에 의해 전사종결이 이루어지지 않는다. SP6 RNA 중합효소는 CGTTTTATCTGTTGTTTG의 서열을 가진 모든 주형에서 전사연장이 중단되었다. 본 연구를 통하여 적어도 세 가지 요소가 이러한 종결신호의 기능에 있어서 필수적임을 규명하였다. 이러한 종류의 신호에 모두 존재하는 ATCTGTT의 DNA 염기서열은 전사종결 위치의 결정에 작용하며 이 염기서열의 돌연변이는 모든 경우 종결신호의 기능을 상실하게 한다. 이 염기서열의 위쪽에 존재하는 주형의 서열도 중요한 요소이다. 이 서열의 변화는 종결효율의 감소 또는 종결신호의 기능상실을 유발할 수 있다. ATCTGTT의 아래쪽에 위치한 우라실이 많은 부위도 전사종결에 있어서 필수적이다. 이 부위는 주형과 전사물 사이의 상호작용의 약화를 유발하여 전사종결을 촉진하는 것으로 보인다. 특정위치에서 전사종결이 이루어지려면 이 부위는 우라실로만 구성되어서는 안된다. 우라실이 연속적으로 존재하면 전사물의 미끌림이 유발된다. SP6 RNA 중합효소와는 대조적으로 T7 중합효소의 효율적인 종결을 위해서는 위의 세 가지 요소 외에도 다른 요소가 필요하다.
