Structural Biochemistry, Reverse Transcription

Introduction

Reverse transcription is the process in which a double stranded DNA molecules are made from a single stranded RNA. The name of this method is formed by its opposite direction to transcription. It also involves the presence of a reverse transcriptase enzyme, a primer, DNTAs and a RNase inhibitor.

Reverse transcriptase

A reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase enzyme that transcribes single-stranded RNA into double-stranded DNA. It also helps in the formation of a double helix DNA once the RNA has been reverse transcribed into a single strand cDNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of this.

Mechanism for a reverse transcription

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Step a: The minus strand known as primer to transfer RNA to form the first DNA strand and to interact with the tRNA 3 end in a polymerase binding mode.

Step b: The enzyme cleaves the RNA template by binding to it in a RNase H mode.

Step c: the reverse transcriptase use the PPT sequence as a primer to bind in the polymerase mode for the synthesis of the second DNA strand.

Retroviruses

Retroviruses store genetic information on RNA. An example of retroviruses are H.I.V. and A.I.D.S.. Retroviruses flow from RNA to DNA. Viruses are enclosed in protein coats and are not capable of independent growth and therefore cannot live without the host.

Although AIDS is a terrible disease which utilizes reverse transcriptase, mankind owes a considerable debt to it. Reverse transcriptase has seen extensive use in the study of gene expression (via gene chips), and protein synthesis (mRNA---reverse transcriptase--->cDNA-->infuse in recombinant plasmid-->insert into E. coli-->have E. coli synthesize more of the original mRNA-->have that mRNA translated into its respective protein).

Retroviral infection. (Image: Harakeli at English Wikibooks, CC BY 3.0)

Disadvantage

Sometimes, the enzyme helping in the method such as reverse transcriptase makes mistakes, leading to the wrong reading of the RNA sequence. It causes in the difference of the single infected cells produced in all viruses. Instead, they form a diversity of molecular differences in their surface coat and enzymes, giving scientists difficulty in inventing the corresponding drug for the disease. As a result, it is difficult to fight HIV with vaccines due to its continual changing in surface molecules.

Invention of drugs for HIV

For a while, reverse transcriptase was considered as a great target for many HIV studies. It is discovered that without reverse transcriptase, the segment of mutated DNA can't become incorporated into the host cell, and therefore, can't be reproduced.

As a result, the very first major class of drugs were found to aim on this enzyme to slow down HIV infections called reverse transcriptase inhibitors. They are: AZT, 3TC, d4T, ddc, and ddl that block the recoding of viral RNA into DNA. Yet, the continual change in HIV surfaces molecules limits the effect of these drugs.

Common HIV drugs and How They Work

There have been quite a few drug therapies starting from the 1990s till 2006, yet still the research for new enhanced drugs are still occurring. Highly Active Antiretroviral Therapy (HAART) consist of three different HIV treatments; Protease Inhibitors, Non-nucleoside reverse transcriptase inhibitor, and Nucleoside reverse transcriptase inhibitor. (Introduction to HAART video HAART)For the Protease Inhibitor (PI) the main target point of this drug is to inhibit the viral protease, which in turn is responsible for Proteolytic processing of the viral polypeptide. There is also a non-nucleoside reverse transcriptase inhibitor (NNRTI) in combination with two nucleoside reverse transcriptase inhibitors (NRTI & NtRTI). NNRTI is a non-competitive inhibitor, which means that it binds to the reverse transcriptase enzyme by binding at a different site. In results in the change of the binding site shape and retarding of the catalyst ability. Relating this to the viral DNA, the movement of protein domains of our target enzyme are stopped. This means that the DNA synthesis doesn't occur. The nucleoside reverse transcriptase inhibitors (NRTI & NTRI) instead work as competitive substrate inhibitors. Competitive inhibitors occur when the substrate competes with the inhibitor at the active site. Relating it to the reverse transcriptase enzyme, we see in the process the deoxynucleotide of the normal DNA competes with the deoxynucleotide aimed towards growing the viral DNA chain. Thus, there is now a 3'-OH group on the deoxyribose unit. This means that deoxyribonucleotide is unable to form the next 5'-3' phosphodiester bond essential for the elongation of the DNA chain. This is called chain termination. (For a general visual of the process NRTI). [1]

Another HIV/AIDS inhibitor is the Diketoaryl (DKA) Integrase inhibitors. Integrase is the 3rd viral enzyme that has a two step catalyses.

1. 3' Processing: The integrase catalyses the processing of the 3'-ends of the viral cDNA. The processing corresponds to an endonucleotlytic cleave of the 3'-ends of the viral cDNA.
2. Strand Transfer: From 3'-processing, the viral 3'-OH cDNA ends are ligated to the 5'-DNA phosphate of an acceptor DNA, which is the host chromsome

Pre-Integration Complex: This macromolecule molecule is formed during and after the 3'-processing which undergoes nuclear translocation. It carries the 3'-processed viral cDNA ends with viral and cellular proteins to the nucleus before integration occurs.
DKA aims to block the stran-transfer step (step 2). The other inhibitors block the strand transfer step and 3'-processing. This integrase inhibitors are still on trial to aid in the future discovery of more specific Anti-AID drugs.[2]

Summary: HIV Inhibitors (Image: Harakeli at English Wikibooks, CC BY 3.0)

List of Current Anti-AID Drugs

Here are a list of current anti-AID therapies that have been approved by the FDA in order of; FDA approval year, brand name, generic name and manufacturer.

Fusion inhibitors
2003 Fuzeon Enfuvirtide (T-20) Roche Pharmaceuticals & Trimeris

Nucleoside reverse transcriptase inhibitors (NRTIs)

1987 Retrovir Zidovudine (AZT) GlaxoSmithKline

1991 Videx Didanosine (ddI) Bristol-Myers Squibb

1992 Hivid Zalcitabine (ddC) Roche Pharmaceuticals

1994 Zerit Stavudine (d4T) Bristol-Myers Squibb

1995 Epivir Lamivudine (3TC) GlaxoSmithKline

1997 Combivir Lamivudine+ Zidovudine GlaxoSmithKline

1998 Ziagen Abacavir GlaxoSmithKline

2000 Trizivir Abacavir + lamivudine + zidovudine GlaxoSmithKline

2000 Videx EC Didanosine (ddI) Bristol-Myers Squibb

2001 Viread Tenofovir disoproxil Gilead Sciences

2003 Emtriva Emtricitabine (FTC) Gilead Sciences

2004 Epzicom Abacavir+ Lamivudine GlaxoSmithKline

2004 Truvada Emtricitabine+ Tenofovir Gilead Sciences

Non-nucleoside reverse transcriptase inhibitors (NNRTIs)

1996 Viramune Nevirapine Boehringer Ingelheim

1997 Rescriptor Delavirdine (DLV) Pfizer

1998 Sustiva Efavirenz Bristol-Myers Squibb

Protease inhibitors (PIs)

1995 Invirase Saquinavir Roche Pharmaceuticals

1996 Norvir Ritonavir Abbott Laboratories

1996 Crixivan Indinavir (IDV) Merck

1997 Viracept Nelfinavir Pfizer

1997 Fortovase Saquinavir Mesylate Roche Pharmaceuticals

1999 Agenerase Amprenavir GlaxoSmithKline

2000 Kaletra Lopinavir+ Ritonavir Abbott Laboratories

2003 Reyataz Atazanavir Bristol-Myers Squibb

2003 Lexiva Fosamprenavir GlaxoSmithKline

[3]

Telomerase Reverse Transcriptase

Understanding the structure of Telomeres is vital for genomic stability. The dysregulation of telomere could lead to Apoptosis(Cell death), and abnormality in the cell proliferation. The enzyme telomerase is essential in the process of maintaining telomere repeats in most eukaryotic cells. This Telomerase consists of a reverse transcriptase enzyme and an RNA strand that controls the synthesis of the G-rich strand of telomere terminal repeats. The telomerase reverse transcriptase contains a particular and variable C- and N- terminal extensions that flank a central domain that is reverse transcriptase like. The telomerase reverse transcriptase has two distinguishable properties which are the stable association with the telomerase RNA and the ability reverse transcribe the RNA segment repeatedly.

In Eukaryotes, telomeres are nucleoproteins located at the end of linear chromosomes. It consists of short sequences in addition to proteins that have interaction with these sequences directly as well as indirectly. One of the telomeres jobs is to protect the chromosome terminal from degradation and other reactions that are inappropriate for the chromosome. It also promotes division of chromosomes during meiosis and mitosis. Incomplete replication of telomeres leads to loss of DNA, which is known as “the end replication problem”. The enzyme Telomerase is very crucial in bacteria cells and is required for the reproduction of cell population. On the other hand, in eukaryotes, the telomerase enzyme is suppressed in normal somatic tissues but highly expressed in the sex tissues such as ovaries and testis. Telomerase is considered a plausible target when it comes to cancer therapy because of its up-regulation in cancer cells. [4]

References

1-3.^ Integrase Inhibitors To Treat HIV/AIDS Yves Pommier, Allison A. Johnson and Christophe Marchand. Volume 4. March 2005.

4. 1Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research,
   Sir Mortimer B. Davis Jewish General Hospital, and Department of Anatomy and Cell
   Biology and Department of Medicine, McGill University, Montreal, Quebec, Canada

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