Initiation transcription

These vectors typically have a polylinker adjacent to the SPG promoter. Successive rounds of transcription initiated by SPG RNA polymerase at its promoter lead to the production of multiple RNA copies of any DNA inserted at the polylinker. Before transcription is initiated, the circular expression vector is linearized by a single cleavage at or near the end of the insert so that transcription terminates at a fixed point. 

Find the translation and transcription initiation sites, find promoter sites, define open reading rames (ORF)... 

The DNA sequence analysis of specific genes has allowed the recognition of a number of sequences important in gene transcription. From the large number of bacterial genes smdied it is possible to construct consensus models of transcription initiation and termination signals. 

Sequences farther upstream from the start site determine how frequently the transcription event occurs. Mutations in these regions reduce the frequency of transcriptional starts tenfold to twentyfold. Typical of these DNA elements are the GC and CAAT boxes, so named because of the DNA sequences involved. As illustrated in Figure 37—7, each of these boxes binds a protein, Spl in the case of the GC box and CTF (or C/EPB,NF1,NFY) by the CAAT box both bind through their distinct DNA binding domains (DBDs). The frequency of transcription initiation is a consequence of these protein-DNA interactions and complex interactions between particular domains of the transcription factors (distinct from the DBD domains—so-called activation domains ADs) of these proteins and the rest of the transcription machinery (RNA polymerase II and the basal factors TFIIA, B, D, E, F). (See... 

A third class of sequence elements can either increase or decrease the rate of transcription initiation of eukaryotic genes. These elements are called either enhancers or repressors (or silencers), depending on which effect they have. They have been found in a variety of locations both upstream and downstream of the transcription start site and even within the transcribed portions of some genes. In contrast to proximal and upstream promoter elements, enhancers and silencers can exert their effects when located hundreds or even thousands of bases away from transcription units located on the same chromosome. Surprisingly, enhancers and silencers can function in an orientation-independent fashion. Literally hundreds of these elements have been described. In some cases, the sequence requirements for binding are rigidly constrained in others, considerable sequence variation is... 

TBP binds to the TATA box in the minor groove of DNA (most transcription factors bind in the major groove) and causes an approximately 100-degree bend or kink of the DNA helix. This bending is thought to facilitate the interaction of TBP-associated factors with other components of the transcription initiation complex and possibly with factors bound to upstream elements. Although defined as a component of class II gene promoters, TBP, by virtue of its association with... 

WUliams SA, Chen LE, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene WC (2006) NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J 25(1) 139-149... 

In Drosophila the two tissue-specific mRNAs are generated by alternative splicing of a single primary transcript (Fig. 9). In vertebrates the two tissue specific AADC transcripts are generated from two alternative promoters (Fig. 11) (Albert et al., 1992 Ichinose et al., 1992 Thai et al., 1993). In neural tissue transcription initiates from exon Nl, whereas in non-neural tissue transcription initiates from exon LI. This produces two distinct primary transcripts that are then spliced from the first exon (LI or Nl) to exon 2 to generate two tissue-specific mRNAs. Translation initiates within exon 2, such that the same AADC protein product is synthesized from both AADC mRNAs. 

Figure 11. Alternate promoters are used for production of the vertebrate neural and non-neural AADC mRNAs. Non-neural AADC transcription initiates at exon L1, whereas neural transcription initiates at exon N1. The non-neural mRNA splices from exon L1 to 2, since the 5 edge of exon N1 is a site of transcriptional initiation instead of a splice acceptor site. Translation initiates from the same AUG in exon 2 in both mRNAs, producing the same protein product in both tissue types. This scheme holds for both human and rat AADC, although the nomenclature of the exons differs. In rat AADC the exon N1 to 2 splice uses a splice acceptor site 5 bp downstream of the splice acceptor used for the exon L1 to 2 splice (Albert et al., 1992).

The highly restricted tissue-specific transcription of c-mos poses an interesting problem in gene regulation during germ cell development. Moreover, c-mos is transcribed from different promoters in mouse spermatocytes and oocytes (Fig. 4) (Propst et al., 1987). In spermatocytes, transcription initiates approximately 280 nucleotides upstream of the c-mos ATG (Propst et al., 1987), whereas the transcription start site in oocytes has been mapped to 53 base pairs upstream of the ATG (Pal et al., 1991). Neither the spermatocyte nor oocyte promoter regions are... 

An additional sequence element within the mouse ZP3 promoter has been identified as the binding site of an oocyte-specific protein called OSP-1 (Schickler et al., 1992), although the possible effect of mutations of this element on transcriptional activity has not been established. Nonetheless, it is interesting that a similar sequence (GATGA) is present 40 nucleotides upstream of the c-mos transcription initiation site (Fig. 5), although deletion of this element had no discernible effect on the activity of the c-mos promoter in microinjected oocytes (Pal et al., 1991). 

It should be noted that the NRE defined in these experiments is distinct from the previously described c-mos UMS sequence (Blair et al., 1984 Wood et al., 1984). The UMS is located approximately 1.4 kb upstream of the c-mos spermatocyte promoter and was identified because it blocked activation of c-mos transforming potential by insertion of retroviral promoters. It is thought to act as a transcriptional terminator, blocking transcription of c-mos initiated at upstream sequences. However, both the spermatocyte and oocyte transcription initiation sites are substantially downstream of the UMS. Moreover, the presence or absence of the UMS does not affect c-mos expression in either microin-jected oocytes (Pal et al., 1991) or transfected NIH 3T3 cells (Zinkel et al., 1992). It thus appears unlikely that the UMS functions as a negative regulator of c-mos transcription from either the spermatocyte or oocyte promoters in somatic cells. 

Pal, S. K., Zinkel, S. S., Kiessling, A. A., and Cooper, G. M. (1991). c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site. Mol. Cell. Biol. 11 5190-5196. 

Paules, R. S., Propst, F Dunn, K. J., Blair, D. G., Kaul, K., Palmer, A. E., and Vande Woude, G. F. (1988). Primate c-mos proto-oncogene structure and expression transcription initiation both upstream and within the gene in a tissue-specific manner. Oncogene 3 59-68. 

Roy, A. L., Meisteremst, M., Pognonec, P., and Roeder, R. G. (1991). Cooperative interaction of an initiator-binding transcription initiation factor and helix-loop-helix activator USF. Nature 354 245-248. 

Mukherjee, S., Brieba, L.G., and Sousa, R. (2002) Structural transitions mediating transcription initiation by T7 RNA polymerase. Cell 110(1), 81-91. 

Beato M, Sanchez-Pacheco A (1996) Interaction of steroid hormone receptors with the transcription initiation complex. Endocr Rev 1 587... 

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