Addition of the fourth nucleotide on the Nascent Transcript: Second Transition

Formation of the third phosphodiester bond creates a 4-nt product. This commits the initiation complex to promoter escape. The short 4-nt transcript is still loosely associated with the RNA polymerase II initiation complex and can dissociate to yield abortive products, which are not further extended. Inhibition of ATP-hydrolysis by TFIIH does not lead to collapse of the open region any longer. The transcription complex has lost the sensitivity to single-stranded oligo-nucleotide inhibition. However, ATP-hydrolysis and TFIIH are required for efficient promoter escape. The open region ("transcription bubble") expands concomitant with the site of RNA-extension. In this case this region spans positions -9 to +4.

第三磷酸二酯键的形成产生了一个4个核苷酸长的产物。这一步骤使得启动复合物得以从启动子区域逸出。这个短暂的4个核苷酸长的转录产物仍然与RNA聚合酶II的启动复合物松散地结合，并可能解离，产生非扩展的终止产物。TFIIH对ATP水解的抑制不再导致开放区域的崩溃。转录复合物对单链寡核苷酸抑制的敏感性已丧失。然而，ATP水解和TFIIH对于有效的启动子逸出是必需的。随着RNA延伸位点的开放区域（转录泡）的扩展，这一区域跨越了从-9到+4的位置。