PART 1
The recombinant protein of the catalytic subunit of human mitochondrial RNA polymerase (h-mtRP) was overexpressed in Escherichia coli., and was purified to near homogeneity. The recombinant h-mtRP shows the same characteristics of the partially purified native enzymes from human mitochondria. The recombinant h-mtRP with recombinant h-mtTFA lacks a promoter-dependent transcription activity, pointing to another component(s) being required for promoter-dependent transcription. The promoter-specific binding of recombinant h-mtRP was observed using the electrophoretic mobility shift assay. The binding affinity of h-mtRP for LSP promoter was much greater than that for the HSP promoter, and was independent to the presence of the mtTFA. The binding of mtTFA to the promoters was also not dependent on the presence of the h-mtRP. The tertiary complexes containing both the mtTFA and h-mtRP bound to promoter sequences were observed. The h-mtRP is shown to possess the novel ability to direct robust but promiscuous transcription with mtTFA on duplex DNA templates with 3’-adenosine protruding ends. Analysis of reaction products indicates that h-mtRP, only with mtTFA, is capable of extending the 3’-termini of duplex DNA having 3’-adenosine-protruding DNA ends by ribonucleotides addition. Based on these findings, it is proposed that h-mtTFA activates the h-mtRP by helping to add the following RNA chains to the 3’-adenosine-terminus of the nascent RNA chain. The missing component for the promoter-directed transcription would be a protein to facilitate the h-mtRP to melt the DNA and to start the initial RNA synthesis.
PART2
Transcription termination of human mitochondrial genome requires the action of a site-specific DNA-binding protein that binds to a single location of the entire human mitochondrial genome. A specific DNA-binding protein (mTERF) for the termination sequence was recently been identified and cloned. To study the functional characteristics of the mTERF, the recombinant protein of mTERF was expressed and purified. The DNA-binding sites for mTERF were selected and amplified by means of a PCR-mediated random-site selection method involving gel-mobility shift analysis. Sequencing and comparison of the selected clones suggested that (G/C)GTGTGGCAGAGCC(G/T/C)GG is the consensus binding sequence, which is similar to the native termination sequence. The heteroduplex-binding assay of mTERF showed that the upper strand is much preferred than the lower strand of the termination sequence and the binding test of the mTERF to the single-stranded DNA shows that the upper strand is also strongly preferred. The effect of the MELAS mutation on mTERF-binding for the single-stranded DNA was much greater than that for the double-stranded DNA of the termination sequence. Another protein, mTLP $(\underline{mT} ERF-\underline{L} ike \underline{P}rotein)$, highly homologous to mTERF, was identified. The mTLP possesses overall sequence homology to mTERF. The cDNA clone of mTLP has some novel features that the polyA signal sequence is in the open reading frame, and the polyA sequence begins within the stop codon, although it is encoded by a nuclear gene. These features are common in mitochondrially encoded genes and may be of evolutionary significance. The mTLP does not bind at all to the transcription termination sequence of human mitochondria.
인체 미토콘드리아 RNA 중합효소를 대장균에서 발현시키고 그 단백질을 순수 정제하였다. 재조합 단백질은 인체 미토콘드리아로 부터 유래한 단백질과 생화학적 특성이 동일하였다. mtTFA와 mtRP만으로는 promoter로 부터 특이적인 RNA가 만들어지지 않아, 또다른 인자가 존재한다는 것을 보여준다. mtRP는 mtTFA와는 독립적으로 promoter와 특이적으로 결합하는 활성을 나타내었다. mtTFA와 mtRP는 동시 존재시, DNA 의 말단에 RNA를 첨가하는 활성을 나타내었다. 이 활성은 mtTFA가 mtRP의 RNA 합성과정중 RNA 첨가반응을 촉진하는 기작을 보여준다고 사료된다.
인체 미토콘드리아의 전사 종결 서열에 결합하는 단백질인 전사 종결 인자 (mTERF)의 결합 염기서열을 SELEX 방법을 이용하여 결정하였다. 그리고 heteroduplex DNA를 이용하여 L-strand가 H-strand보다 결합에 더 중요하다는 것을 관찰 하였고, 외가닥 결합에도 L-strand가 선택적으로 결합되는 것을 관찰하였다. MELAS 유전병을 일으키는 돌연변이로 인한 mTERF 의 결합은 이중나선인 경우 보다 외가닥 결합시 더 큰 손상을 가져왔다.
mTERF와 유사한 새로운 mTLP를 발견하였다. mTLP는 mTERF와는 달리 전사 종결 서열에 결합하는 활성은 관찰되지 않았다. mTLP는 핵내 유전자임에도 불구하고, 그 구조를 보면 polyA 신호가 단백질 코딩 부위내에 포함되어 있고, 단백질 합성 종결 신호가 polyA에 의해서 일어나고, intron을 가지지 않는 전형적인 미토콘드리아 내의 유전자들의 특징을 그대로 간직하고 있어, 진화적으로 매우 중요하게 보인다.
