RNA Conformation in the Tat-TAR Complex Determined by Site-Specific Photo-Cross-Linking

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• 作者：Zhuying Wang and Tariq M. Rana
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：May 21, 1996
• 年：1996
• 卷：35
• 期：20
• 页码：6491 - 6499
• 全文大小：415K
• 年卷期：v.35,no.20(May 21, 1996)
• ISSN：1520-4995

文摘

Transcriptional regulation in human immunodeficiency virus type 1(HIV-1) requires specificinteractions of Tat protein with the transactivation responsive region(TAR) RNA, a 59-base stem-loopstructure located at the 5'-end of all mRNAs. We have used asite-specific cross-linking method basedon 4-thiouracil (4-thioU) photochemistry to determine the conformationof TAR RNA and its interactionwith Tat protein under physiological conditions. Three differentTAR RNA constructs with a single4-thioU residue at position 23, 38, or 40 were synthesized. UponUV irradiation, 4-thioU at all threepositions formed interstrand covalent cross-links in TAR RNA.Determination of cross-link sites by RNAsequencing revealed that 4-thioU at position 23 makes a direct contactwith U40, while a 4-thioU atposition 40 cross-links to C24 and C25, and at position 38, 4-thioUcontacts G26 in TAR RNA. Theaddition of arginine did not alter the yield or the site of RNA-RNAcross-links. However, in the presenceof Tat(38-72), UV irradiation of RNA modified with 4-thioU atposition 23 or 38 resulted in RNA-protein cross-links, but no RNA-RNA cross-links were observed.4-thioU at position 40 formed bothRNA-RNA and RNA-protein cross-links in the presence ofTat(38-72). An intriguing finding of ourstudies was that a cross-linked TAR RNA with 4-thioU at position 40retained specific Tat-binding activity.Our results establish four important conclusions about Tat-TARstructure. (1) U23 of free TAR RNAis in close contact with U40. (2) U40 is in close proximity to C24and C25 both in free TAR RNA andin a complex with Tat. (3) Tat protein directly contacts U23, U38,and U40 in the major groove of TARRNA. (4) Tat protein can recognize a TAR RNA structure containingan interrupted bulge which isformed by a covalent link between U40 and two bulge residues, C24 andC25. These structural studiesprovide new insights into tertiary folding of TAR RNA and itsinteraction with Tat protein.