M4 RNA Pol II genes: promoters and enhancers -----RNA Polymerase II (RNA Pol II) is located in the nucleoplasm. It is responsible for the transcription of all protein-coding genes and some small nuclear RNA genes. The pre-mRNAs must be processed after synthesis by cap formation at the 5’-end of the RNA and poly (A) addition at the 3’-end, as well as removal of introns by splicing. Molecular Biology

TATA box Transcription Transcriptional start site DNA Coding-strand sequences: TATAAAA GC + CAAT boxes –100 –50–25 +1 Py 2 CAPy 5 Promoters TATA box: Many eukaryotic promoters contain a sequence called the TATA box around bp upstream from the start site of transcription. It has the 7 bp consensus sequence 5’-TATA （ A/T ） A （ A/T ） -3’ although it is now known that the protein which binds to the TATA box, TBP, binds to an 8 bp sequence that includes an additional downstream base pair, whose identity is not important. Initiator element: The initiator element is located around the transcription strt site. Many initiator elements have a C at -1 and A at +1. Molecular Biology

Upstream regulatory elements These elements are found in many genes which vary widely in their levels of expression in different tissues. Two common examples are SP1 box, which is found upstream of many genes both with and without TATA boxes, and the CCAAT box. Promoters may have one, both or multiple copies of these sequences. These sequences which are often located within bp upstream from the promoter are referred to as upstream regultory elements (UREs) and play an important role in ensuring efficient transcription from the promoter. Molecular Biology

Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site. This kind of elements are called as enhancer. Classically, enhancers have the following general chracteristics: They exert strong activation of transcription of a ;linked gene from the correct start site. They activate transcription when placed in either orientation with respect to linked genes. They are able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start site. They exert preferential stimulation of the closest of two tandem promoters. Enhancers Molecular Biology

M5 General transcription factors and RNA Pol II initiation RNA Pol II basal transcription fators ： A serial of nuclear transcription factors have been identified, purified amd cloned. These are required for basal trancription initiation from RNA Pol II promoter sequences in vitro and named as TFIIA ， TFIIB ， TFIIC ， TFIID. They have been shown to assemble on basal promoters in a specific order and they may be subject to multiple levels of regulation. TFIID ： Inn promoters containing a TATA box, the RNA Pol II transcription factor TFIID is responsible for binding to this key promoter element. The binding of TFIID to the TATA box is the earliest stage in the formation of the RNA Pol II transcription initiation complex. It seems that in mammalian cells, TBP binds to the TATA box and is then joined by at least eight 由 TBP 和 TAF IIs to form TFIID. Molecular Biology

TBP TBP is present in all three enkaryotic transcription complexes and clearly plays a major role in transcription initiation. TBP is a monomeric protein, with a highly conserved C-terminal domains of 180 residues and this conserved domain functions as well as the full-length protein in in vivo transcription. Molecular Biology

TBP structure TBP has been shown to have saddle structure with an overall dyad symmetry, but two halves of the molecule are not identical. TBP interacts with DNA in the minor groove so that the inside of the saddle binds to DNA at the TATA box and the outside surface of the protein is available for interactions with other protein factors. Binding of TBP deforms the DNA so that it is bent into the inside of the saddle unwound. This results in a kink of about 45° between the first two and last two base pariss of the 8 bp TATA element. Molecular Biology

TFIIA, TFIIB and RNA polymearse binding TFIIA: TFIIA binds to TFIID and enhances TFIID binding to the TATA box, stabilizing the TFIID-DNA complex. TFIIA is made up of at least three subunits. TFIIB and RNA polymearse binding: Once TFIID has bound to the DNA, another transcription factor, TFIIB, binds to TFIID. TFIIB can also bind to the RNA polymerase. This seems to be an important step in transcription initiation since TFIIB asts as a bridging fator allowing recruitment of the polymerase to the complex togather with a further fator, TFIIF. Molecular Biology

Factors binding after RNA polymerase After RNA polymerase binding, three other transcription factors, TFIIE, TFIIH, and TFIIJ, rapidly asociate with the compex. These proteins are necessary for transcription in vitro and associate with the complex in a defined order. TFIIH is a large compex which is made up of at least five subunits. TFIIJ remains to fully characterized. Molecular Biology

CTD phosphorylation by TFIIH TFIIH is a large multicomponent protein compex which contains both kinase and helicase activity. Activation of TFIIH results in phophorylation of the carboxyl-terminal domain (CTD) of the RNA polymerase. This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region. TFIIH therefore seems to have a very important function in control of transcriptiom elongation. Molecular Biology

TFIID TFIIB TFIID TFIIF TATA box TFIIB binds to TFIID. TFIIB acts as a bridge to bind to RNA polymerase II/TFIIF. TFIIE and TFIIH bind to RNA polymerase II. RNA polymerase TFIIB TFIIA TFIIA binds to TFIID and enhances TFIID binding to TATA box. Once TFIID has bound to the DNA ， TFIIB binds to TFIID. Molecular Biology

After RNA polymerase binding, three other transcription factors, TFIIE, TFIIH, and TFIIJ, rapidly asociate with the compex Activation of TFIIH results in phophorylation of the carboxyl- terminal domain (CTD) of the RNA polymerase. This phosphorylation results on formation of a processive RNA polymerase complex and allows the RNA polymerase to leave the promoter region. Transcription factors Molecular Biology

The initiator transcription complex Many RNA Pol II promoters which do not contain a TATA box have an initiator element overlapping their start site. It seems that at these promoters TBP is recruited to the promoter by a further DNA-binding protein which binds to the initiator element. TBP the recruits the other transcription factors and RNA polymerase in a manner similar to that which occurs in TATA box promoters. Molecular Biology

Vmax = 50 nts / sec  About 1/10th DNA polymerase  Which needs to be fast:  Initiated at very few points  Only ~ 10 DNA pol molecules / cell  Each has to be very fast  ~3,000 molecules / cell  Transcription simultaneously at many points RNA pol. Speed Molecular Biology

Error rate of ~ to  Much greater than DNA pol  Less than expected just from W.-C base-pairing  Suggests proof-reading Details of proof-reading not understood. RNA pol. Fidelity Molecular Biology

What about the dsDNA sequence tells RNA polymerase where to start? Be sure to be able to distinguish between prokaryotes and eukaryotes in your answer. Your homework Molecular Biology