Reverse transcriptase
Reverse transcriptase (RT) or RNA-dependent DNA polymerase transcribes single-stranded RNA into double-stranded DNA. HIV-1 RT is from the human immunodeficiency virus and is a heterodimer of P66 and P51. The images at the left and at the right correspond to one representative RT structure, i.e. crystal structure of HIV-1 Reverse transcriptase (1hmv). P15 is its RNAse H domain. NNRTI are the non-nucleoside inhibitors of HIV-1 RT. M-MLV is RT from the leukemia virus. Being the protein that gives their name to Retroviruses, Reverse Transcriptase is, in company of Protease and Integrase, the most important part of the protein system involved in the process of infection of viruses like HIV, MuLV and AMV, and has the unusual property of transcribing ssRNA into dsDNA going against the Central Dogma of Molecular Biology. Since its discovery in 1970, the study of its properties and mechanisms of action have been of high interest among the scientific community due to the unique properties that makes it an important medical target enzyme and important tool for genetic engineering applications like RT-PCR in the construction of cDNA libraries.
StructureStructure
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Reverse Transcriptase is one of the CBI Molecules being studied in the University of Massachusetts Amherst Chemistry-Biology Interface Program at UMass Amherst and on display at the Molecular Playground.
This hand-like protein that has an usual length of 1000 residues (560 in Chain A and 440 for B), the third of them involved in alpha helical and almost a quarter in beta sheets, showing α+β domains; has an usual weight of 66KDa whereas is around 51KDa, those monomers are derived from the same gen but p51 lacks the aminoacids of one active site and has a different tertiary structure conformation compared with p66, for this reason is totally inactive. [1]
Template:STRUCTURE 3hvt
FunctionFunction
As a RNA-dependent DNA Polymerase, is able to recognize the initial RNA, transcribe it to ssDNA, cleave the remaining RNA and then build up the dsDNA, to do this the protein has two active catalytic zones. Chain A has the that consist of two finger-like domains, one of them recognizes the initial nucleic acid by h-bonds interactions with phosphate groups of the side chains, then both domains make a conformational change closing the recognition hole to allow the second domain with the support a coordination system, begin the transcription process adding the specific DNA nucleotides; this change is allowed by a between the two previous domains that is used as a common pharmaceutical target site in order to prevent this change and by this way inhibit the activity, but this one is the only zone of the Chain A that has non-conserved aminoacids giving the virus more drug resistance. [2] As the same rate that the polymerization process occurs, the other active site known as the , cleaves the RNA releasing the ssDNA that comes again trough the Polymerase active site to become dsDNA (all this with a coordinative system, that allows non-specific recognition, just with phosphates). Finally Chain B despite of the similar aminoacid sequence with Chain A has no enzyme activity, possibly its function is to stabilize and communicate both active sites, this varying the length between them in order to synchronize both functions. This seems to be the most general idea of the mechanism of action of Reverse Transcriptase, however the process remains unclear and new approaches are being reported. [3]
One of the principal issues about this protein comparative with the usual DNA polymerase (besides to the similarity with the Klenow fragment), is the lack of a correction mechanism (usually made by DNA PolIII in the DNA Polymerase) that increase the quota of errors, producing more mutations and by this way giving more facultative and resistance abilities to the virus.
3D Structures of Reverse transcriptase3D Structures of Reverse transcriptase
HIV-1 RTHIV-1 RT
3dlk, 1hmv - P66/P51 1mu2, 1jle, 1hpz, 1hqu, 1qe1, 1dlo - P66/P51 (mutant) 1har - P66 N-terminal 3lp0, 3lp1, 3lp2, 1vrt - P66/P51+NNRTI nevirapine 2hnd, 2hny, 1s1u, 1s1x, 1lw0, 1lwc, 1lwe, 1lwf, 1jlb, 1jlf, 1fkp - P66/P51 (mutant)+NNRTI nevirapine 3m8p, 3mec - P66/P51+NNRTI etravirine 3med - P66/P51 (mutant)+NNRTI etravirine 3mee, 2zd1- P66/P51+NNRTI rilpivirine 3meg, 2ze2, 3bgr - P66/P51 (mutant)+NNRTI rilpivirine 1ikw, 1fk9 - P66/P51 +NNRTI efavirenz 1jkh, 1ikv, 1fko - P66/P51 (mutant)+NNRTI efavirenz 3dya, 3e01, 3drp - P66/P51+NNRTI pyrazole 3drr, 3drs - P66/P51 (mutant)+NNRTI pyrazole 3irx, 3is9, 3lak, 3lal, 3lam, 3lan, 3ffi, 3di6, 3dle, 3dlg, 2rki, 3c6t, 3c6u, 2rf2, 2vg5, 2vg6, 2vg7, 2opp, 1jlq, 1ep4, 1c0t, 1c0u, 1dtt, 1dtq, 1bqm, 1rt1, 1rt2, 1rev, 1rtj, 1vru, 1hnv, 1hni - P66/P51+NNRTI 1s1t, 1s1v, 1s1w, 1s6p, 1s6q, 1s9e, 1s9g, 1suq, 1sv5, 3dm2, 3dmj, 3dok, 3dol, 2opq, 2opr, 2ops, 2ic3, 2hnz, 1jlg, 1bqn, 1uwb, 1tvr - P66/P51 (mutant)+NNRTI 3kk1, 1c1b, 1rti - P66/P51+nucleotide inhibitor 1jla - P66/P51 (mutant)+ nucleotide inhibitor 3ig1 - P66/P51+ inhibitor beta-thujaplicinol 3i0r, 3i0s, 3m8q, 3isn, 3ith, 1c1c - P66/P51+ pyrimidine derivative 1ikx - P66/P51 (mutant)+ pyrimidine derivative 1tv6 - P66/P51+ pyridine derivative 1jlc, 1iky, 1eet - P66/P51(mutant)+ pyridine derivative 2b5j, 2ban, 2be2, 1rth, 1hvt - P66/P51+ pyridone derivative 1lw2, 1rt3 - P66/P51 (mutant)+ pyridone derivative 1tkt, 1tkx, 1tkz, 1tl1, 1tl3 - P66/P51+ quinoline derivative 2b6a - P66/P51+ THR-50 2i5j - P66/P51 (mutant)+hydrazone derivative 1rt4, 1rt5, 1rt6, 1rt7 - P66/P51+ carboxanilide derivative 1klm - P66/P51+piperazine derivative 2jle – P66+NNRTI 3kjv, 3kk3, 1r0a, 1n5y, 1n6q, 1hys - P66/P51+DNA 1j5o, 1rtd - P66/P51 (mutant)+DNA 1hvu - P66/P51+RNA 1t03, 1t05 - P66/P51+DNA+Fab+tenofovir 2hmi - P66/P51 (mutant)+DNA+Fab 2iaj - P66/P51 (mutant)+ATP 3kk2 - P66/P51+DNA+ATP 3jyt - P66/P51 (mutant)+DNA+ATP 1rdh, 1hrh – P15 3lp3 – P15+inhibitor MK3 3k2p – P15+inhibitor beta-thujaplicinol 3hyf – P15+pyrimidine derivative 1o1w – P15+Mg - NMR
M-MLVM-MLV
2hb5, 1mml - catalytic fragment 2r2r, 2r2t, 2fjv, 2fjw – catalytic fragment+DNA 2r2s, 2r2u - catalytic fragment+DNA+Co Bleomycin 2fjx, 1ztw - catalytic fragment+DNA 1ztt - catalytic fragment+DNA+netropsin 1rw3, 1nnd – fragment (mutant) 1i6j, 1d0e, 1d1u, 1qaj, 1qai – N-terminal+DNA
See AlsoSee Also
- Reverse Transcriptase at Wikipedia
- Molecule of the Month (09/2002) at RCSB Protein Data Bank
- List of Reverse Transcriptase articles at Proteopedia and at RCSB Protein Data Bank
- of Reverse Transcriptase as one of the CBI Molecules on the Molecular Playground
ReferencesReferences
- [1] Kohistaedt, L.A. et al. Science. 256, 1783-1790 (1992) | doi:10.1126/science.1377403
- [2] Consurf Server Data Base. Evolutionary conservation profile for Reverse Transcriptase PDB file 1JLB
- [3] Abbondanzieri, E.A. et al. Nature 453, 184-189 (2008) | doi:10.1038/nature06941