Product Focus: TNT^® Coupled Reticulocyte Lysate System

Functional requirement of p23 and Hsp90 in telomerase complexes. 

Telomeres are specialized structures at the ends of linear chromosomes that in conjunction with the telomerase enzyme provide a mechanism to maintain chromosome length. Vertebrate telomeres are composed of many kilobases of TTAGGG repeats. The enzyme telomerase is a ribonucleoprotein that uses the complementary sequence in its associated RNA component as a template for a reverse transcription addition reaction that adds telomeric DNA to the chromosome ends. The catalytic core of the human telomerase enzyme consists of a reverse transcriptase (hTERT) complexed with the RNA template component (hTR). Native telomerase migrates as a very high molecular weight complex on glycerol gradients, suggesting it exists in a complex with a variety of other cellular proteins. To identify telomerase-associated proteins that may contribute to the assembly and function of the holoenzyme, the authors used the yeast two-hybrid system and verified the requirement of hTERT-associated proteins in the assembly of active telomerase in vitro and in vivo. The authors demonstrated that the presence of rabbit reticulocyte lysate resulted in a significant enhancement of detectable telomerase activity. They further identified the molecular chaperones p23 and Hsp90 as the proteins that bind to the catalytic subunit of telomerase, and found that a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. TNT^® Coupled Reticulocyte Lysate System^(a,b,c) (Cat.# L4600 series) was used for the in vitro expression of hTERT.

Holt, S.E.^1,2, Aisner, D.L.¹, Baur, J.¹, Tesmer, V.M.¹, Dy, M.¹, Ouellette, M.¹, Trager, J.B.³, Morin, G.B.³, Toft, D.O.⁴, Shay, J.W.¹, Wright, W.E.¹ and White, M.A.¹ (1999) Genes Dev. 13, 817.

¹Department of Cell Biology and Neuroscience, University of Texas (UT) Southwestern Medical Center, Dallas, TX  75235; ²Department of Pathology and Human Genetics, Medical College of Virginia/Virginia Commonwealth University, Richmond, VA  23998; ³Geron Corporation, Menlo Park, CA 94025; ⁴Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, MN 55905 USA

Product Focus: TNT^® Coupled Reticulocyte Lysate System

Telomerase RNA function in recombinant Tetrahymena telomerase.

Telomerase is a ribonucleoprotein reverse transcriptase that adds simple telomeric repeat sequences to chromosome ends in vivo as well as to single-stranded primers in vitro. The enzyme is responsible for maintaining chromosome length, and in some cell types, telomerase activity at telomeres regulates cellular proliferative life span. The authors investigated the sequences and structures of recombinant Tetrahymena thermophila telomerase RNA necessary for physical association and activity with the telomerase catalytic subunit expressed in rabbit reticulocyte lysate. They found that phylogenetically conserved primary sequences and a nonconserved secondary structure are essential for telomerase RNA function. Telomerase RNA binding to the catalytic subunit requires sequences 5´ of the template and is highly sequence specific. They also demonstrated that the production of active recombinant telomerase requires a factor in rabbit reticulocyte lysate that promotes ribonucleoprotein assembly. TNT^® Coupled Reticulocyte Lysate System^(a,b,c) (Cat.# L4600 series) was used to express recombinant epitope-tagged telomerase in vitro.

Licht, J.D. and Collins, K. (1999) Genes Dev. 13, 1116.

Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA  94720 USA

Product Focus: Luciferase Assay System

Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.

Survival factors suppress the intrinsic cell death machinery and prevent apoptosis, a naturally occurring programmed cell death process that is crucial for development and maintenance of an organism. Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, such as BAD and caspase-9. This work demonstrates that Akt also regulates the activity of FKHRL1, a member of the human family of Forkhead transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to the association of this protein with other cellular proteins (14-3-3) and retention in the cytoplasm. Factor withdrawal results in dephosphorylation of FKHRL1 nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most probably by inducing the expression of genes critical for apoptosis. The Luciferase Assay System^(a) (Cat.# E1500) was used to measure expression of a FHRE (Forkhead responsive element) luciferase reporter vector in the absence and presence of Akt-induced phosphorylation of FKHRL1.

Brunet, A.¹, Bonni, A.¹, Zigmond, M.J.¹, Lin, M.Z.¹, Juo, P.², Hu, L.S.¹, Anderson, M.J.³, Arden, K.C.³, Blenis, J.² and Greenberg, M.E.¹ (1999) Cell 96, 857.

¹Division of Neuroscience, Children's Hospital and Department of Neurobiology, Harvard Medical School, Boston, MA  02115; ²Department of Cell Biology, Harvard Medical School, Boston, MA  02115; ³Ludwig Institute for Cancer Research, University of California-San Diego, San Diego, CA  92093 USA

^(a)U.S. Pat. No. 5,283,179 and other patents. Certain applications of this product may require licenses from others.

^(b)U.S. Pat. Nos. 5,492,817 and 5,665,563 and other patents.

^(c)The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673.

TNT is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office.

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