Structural Studies Of The C-myc Gene Regulation

人c-myc原癌基因的转录受启动子上游和下游多个顺式元件的调控。c-myc启动子上游的一个顺式元件是CT元件。这是位于P1启动子上游100个碱基的富含CT的序列。结合CT元件的编码链的一种蛋白质是hnRNPk。hnRNPk与CT元件的结合上调c-myc转录。在hnRNPk中有三个同源重复序列，称为KH结构域。hnRNPk蛋白的86个残基的C-末端区段包含hnRNPk中的第三个KH基序（KH 3）。除了常规的蛋白质NMR方法之外，还通过使用液晶技术的NMR光谱测定了其三维结构。液晶环境在系统中产生轻微的秩序，其重新引入偶极耦合，提供有用的结构信息。使用偶极耦合信息的添加计算的结构的最终族导致0.17埃的族的均方根偏差（RMSD）。相比之下，在没有偶极耦合的情况下进行的计算具有0.32埃的RMSD。这些提供了结构精度的定量评估，其中较小的RMSD值意味着较高的精度。这种比较是在理想条件下进行的：所有NMR实验的信噪比都很好，一些NMR实验重复两次以进行误差估计和一致性检查，所有距离估计都是保守进行的，以考虑任何可能的系统误差。因此，对于非理想条件下的典型NMR结构，随着偶极耦合信息的增加，RMSD值的增加应该大得多。在平行的KH 3结构的测定，我们还开发了一个简单的协议的结构细化使用偶极耦合信息。该方案提供给任何希望利用偶极耦合信息的实验室。对KH 3结构域进行了动力学研究。在经历快速（ps）波动的KH 3结构域中鉴定出一个柔性环（L52-R56）。与先前对FMR 1的类似KH结构域的研究相反，保守环（G30-G33）不显示任何灵活性。为了更好地了解c-myc通过CT元件的调节，已经启动了一个平行的项目，以确定细胞核酸结合蛋白（CNBP）的结构。该蛋白结合hnRNPk结合位点对面的链。CNBP上调CT元件活性。构建了全长（176个残基）CNBP。它由七个锌指组成。正在探索仍然保留核苷酸结合亲和力的CNBP的最小构建体。我们完成了突变型（Gly 26-Arg）KH_3、野生型KH_3和KH_3 +ss-DNA复合物的NMR主链动力学研究。我们已经证明突变体KH 3没有ss-DNA结合活性。我们还对KH 3 +DNA复合物进行了滴定研究，以绘制DNA结合位点。在这一点上，我们可以访问所有的动态参数和DNA结合位点的地图。我们目前正在比较所有这些参数，以表征KH 3 ss-DNA相互作用的基础上的结构以及动态信息。我们还启动了侧链动力学的研究，使用KH 3作为模型系统。我们已经开发了一个实验，我们可以探测谷氨酰胺和天冬酰胺侧链上的NH 2部分。该NH 2基团在游离和结合形式下的动力学比较将提供关于侧链与ss-DNA靶标相互作用的信息。这种方法将被扩展到看看CH，CH 2和CH 3部分的蛋白质。去年，我们一直在尝试进行实验，以确定含KH 3的蛋白质中的结构域间运动，并完成我们关于hnRNPk的KH 3侧链运动的项目。为了研究域间运动，我们使用了一个模型系统，已被广泛研究，以测试我们的分析模型。选择的模型系统是钙结合蛋白，钙调素。我们已经能够证实，为了量化慢域间运动的一系列NMR骨干弛豫数据在不同的场强将是必需的。此外，我们的简单模型，代表了一阶的方法来解决这个问题，似乎是足够的，以提供这种运动的幅度以及时间尺度。通过对钙调蛋白中的域运动进行空间建模，已经证实了估计的运动幅度。我们现在应用这个简单的运动模型来研究KH 3的运动。 我们改进了可用于描述蛋白质慢内部结构域运动的运动模型。我们的最新模型使用了Pade近似的摆动在一个圆锥，这导致在一个描述的内部运动的三个指数项。到目前为止，我们的测试已经表明，当运动较大时（弯曲角度为50度或更大），该模型是更合适的模型。在小振幅慢运动的限制下，早期的和数学上更简单的模型是足够的。

The transcription of the human c-myc proto-oncogene is regulated by multiple cis-elements upstream as well as downstream of the promoter sites. One cis-element upstream from the c-myc promoters is the CT element. This is a CT rich sequence which is located 100 bases upstream from the P1 promoter. One protein that binds the coding strand of the CT element is hnRNPk. Binding of hnRNPk to the CT element upregulates c-myc transcription. There are three homology repeats in hnRNPk called the KH domains. The 86 residue C-terminal segment of the hnRNPk protein comprises the third KH motif (KH3) in hnRNPk. The three-dimensional structure of this has been determined by NMR spectroscopy using the liquid crystal technique in addition to the conventional protein NMR method. The liquid crystal environment produces a slight order in the system which reintroduces dipolar coupling providing useful structural information. The final family of structures calculated using the addition of dipolar coupling information results in root mean square deviation (RMSD) for the family of 0.17 Angstrom. In contrast, calculations carried out without the dipolar couplings has an RMSD of 0.32 Angstrom. These provide a quantitative evaluation of the precision of the structures with the smaller RMSD value implying higher precision. This comparison was made under ideal conditions: good signal to noise for all of the NMR experiments, some NMR experiments repeated twice for error estimates as well as consistency check, and all distance estimates done conservatively to take into account any possible systematic error. Thus, for a typical NMR structure under non-ideal conditions, the increase of the RMSD value with the addition of dipolar coupling information should be much greater. In parallel to the KH3 structure determination we also developed a straight forward protocol of structure refinement using the dipolar coupling information. This protocol is being provided to any laboratory which wishes to take advantage of dipolar coupling information. A dynamic study of KH3 domain has also been carried out. One flexible loop (L52-R56) was identified in the KH3 domain which undergoes rapid (ps) fluctuation. In contrast to a previous study of a similar KH domain of FMR1 the conserved loop (G30-G33) does not show any flexibility. In order to get a better understanding of c-myc regulation through the CT element, a parallel project has been initiated to determine the structure of the cellular nucleic acid binding protein (CNBP). This protein binds the strand opposite to the hnRNPk binding site. CNBP upregulates the CT element activity. A construct of the full length (176 residues) CNBP has been made. It consists of seven zinc fingers. A minimum construct of CNBP which still retains the nucleotide binding affinity is being probed. We have completed the NMR backbone dynamic studies of mutant (Gly26-Arg) KH3, wild type KH3, and KH3+ss-DNA complex. We have shown that there is no ss-DNA binding activity for the mutant KH3. We have also done titration studies on the KH3+DNA complex to map the DNA binding site. At this point, we have access to all dynamic parameters and maps of the DNA binding site. We are currently comparing all of these parameters to characterize the KH3 ss-DNA interaction based on structure as well as dynamic information. We also have initiated a study on side chain dynamics using KH3 as a model system. We have developed an experiment where we can probe the NH2 moiety on the side chain of Gln and Asn. Comparison of the dynamics of this NH2 group in the free and bound form would provide information on side chain interaction with the ss-DNA target. This methodology will be extended to look at CH, CH2, and CH3 moieties in the protein. This last year we have been trying to carry out experiments to identify inter-domain motions in KH3-containing protein as well as to finish our project on the side chain motion in KH3 of hnRNPk. In order to study the inter-domain motions we have used a model system that has been studied extensively to test our analysis model. The model system chosen was a calcium binding protein, calmodulin. We have been able to confirm that in order to quantify slow inter-domain motions a series of NMR backbone relaxation data obtained at different field strengths would be required. Furthermore, our simple model that represents a first order approach to the problem, seems to be adequate to provide the amplitude as well as the time scale of this motion. The estimated amplitude of motion has been confirmed by sterically modeling the domain motion in calmodulin. We are now applying this simple motional model to study KH3 motion. We have refined the motional model that can be used to describe slow internam domain motions in protein. Our latest model use a Pade approximation of wobbled in a cone which results in a description of the internal motion by three exponential terms. So far our tests have shown this model to be a more suitable model when the motion is large (angle of inflection of 50 degrees or larger). In the limit of small amplitude slow motion the earlier and mathematically simpler model is sufficient.