Sandbox Reserved 651

This Sandbox is Reserved from 30/08/2012, through 01/02/2013 for use in the course "Proteins and Molecular Mechanisms" taught by Robert B. Rose at the North Carolina State University, Raleigh, NC USA. This reservation includes Sandbox Reserved 636 through Sandbox Reserved 685.

To get started: Click the edit this page tab at the top. Save the page after each step, then edit it again. Click the 3D button (when editing, above the wikitext box) to insert Jmol. show the Scene authoring tools, create a molecular scene, and save it. Copy the green link into the page. Add a description of your scene. Use the buttons above the wikitext box for bold, italics, links, headlines, etc. More help: Help:Editing For more help, look at this link: http://proteopedia.org/w/Help:Getting_Started_in_Proteopedia Human Immunodeficiency Virus-1 Reverse TranscriptaseHuman Immunodeficiency Virus-1 Reverse Transcriptase IntroductionIntroduction The retrovirus human immunodeficiency virus (HIV) is a subsequent progression to acquired immune deficiency syndrome (AIDS). This disease is a continuous worldwide epidemic. The protein HIV-1 reverse transcriptase is one of the key players in the mechanism of infections by this retrovirus. The protein HIV-I reverse transcriptase is the enzyme that’s main responsibility is to copy a single-stranded viral RNA genome into a double stranded DNA.In turn the newly developed DNA can then be incorporated into the host cell genome. The HIV-1 reverse transcriptase enzyme has two domains within its structure. The two domains are a DNA polymerase domain and ribonuclease H (also called RNase H) domain. The role of the DNA polymerase is to copy either RNA or DNA template strands. The purpose of the ribonuclease H is to cleave the RNA duplex after the DNA synthesis has occurred so that the newly created DNA can generate a second strand. StructureStructure spinqualitylabelsall models HIV-1 RT in complex with efavirenz Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image HIV Reverse Transcriptase consists of two subunits; p66 and p51. The subunit p66 is larger and resembles the human right hand in which it can be split into sub domains. The RNase H domain is only found in the p66 subunit and it is responsible for chewing up the RNA template strand after DNA has been synthesized. The 3-D structure of RNase H consists of a five-stranded beta-sheet surrounded by a distribution of alpha helices. However, RNase H that is found in HIV-Reverse Transcriptase the enzyme is missing one of the helices. This helix is known as the C-helix. Since RNase H does not appear to recognize specific sequences, this raises a question of how the enzyme is able to cleave these substrates specifically. Although we do not have a complete answer, we do have the structure of HIV-1 RT in a complex with an RNA/DNA duplex that contains the polypurine tract (PPT) primer. This structure shows that there are unpaired and mispaired bases in the PPT. The structure also shows where there are contacts between amino acids in the RT and the RNA/DNA substrate. Based on the contacts seen in the structure of the RT/RNA/DNA complex, we made mutations in amino acids in the RNase H that contact either the RNA or the DNA strand. Analysis of the cleavages made by RNase H, both in vitro and in vivo, makes it possible to ascertain which contacts are the most important in determining the specificity of RNase H cleavage. The DNA Polymerase in its crystal structure is folded into an open conformation containing the polymerase . The available crystal structures provide useful insights into the relationship between the movements of RT and the discreet steps in the polymerization reaction. The DNA polymerase exhibits the same characteristics in both the p66 and p51 subunits. Yet, the spatial arrangement of the DNA polymerase is different between each subunit. MechanismMechanism HIV-RT is a multifunctional enzyme, possessing three separate activities. The first being the RNA dependent polymerase(RDDP) where the enzyme makes a single strand copy of DNA using the viral RNA as the template strand. With the completion of the RDDP step a RNA-DNA duplex is formed and it is at this stage the RNase H domain of the enzyme facilitates RNA release from the cDNA strand. Once the cDNA has been released the enzyme enters its DNA dependent DNA polymerase(DDDP) activity. During this phase, the double strand DNA copy of the viral genome is completed and is ready for incorporation into the host cells DNA. It is also important to note that HIV-RT has an unusually high error rate for a polymerase enzyme. During the DDDP activity HIV-RT can have an error rate as great as 1 in every 5900 nucleotides added to the developing chain. Inhibition and Drug DesignInhibition and Drug Design The reverse transcriptase enzyme is on of several targets for inhibition in the effort to fight HIV infection. Inhibitors for HIV RT can be grouped into two distinct classes; nucleotide RT inhibitors (NRTIs) and non-nucleotide RT inhibitors (NNRTIs). Nucleotide RT Inhibitors NRTIs function through a competitive inhibition mechanism. These molecules are phosphorylated by cellular kinases and as a result mimic the structure of nucleotides. However, unlike actual nucleotides, NRTIs do not posses the 3'-OH group and as a result terminate chain elongation once they are incorporated into the DNA strand by the enzyme. Non-nucleotide RT Inhibitors NNRTI's are non-competitive inhibitors that bind to a specific site on the enzyme but not to the active site. These molecules are typically hydrophobic and bind to a hydrophobic pocket on RT that is in close proximity to the active site. There are some NNRTIs that have been found that do not follow this general scheme and bind to alternative locations on the enzyme, these locations vary with each inhibitor. NNRTIs are highly specific and rarely have any effect on more than one strain of HIV. ReferencesReferences