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Although the RT structures from [[human]], [[murine]] and [[Bird|avian]] [[retroviruses]] display different subunits, the relative sizes, orientation and connection of the DNA polymerase and RNase H domains are strikingly similar. The RNase H domain occupies ~25% of the RT protein [[C-terminal]]. The DNA polymerase domain occupies ~55% of the RT protein N-terminal.<ref>{{cite journal|last=Beilhartz|first=GL|author2=Götte |title=HIV-1 ribonuclease H: structure, catalytic mechanism and inhibitors.|journal=Viruses|year=2010|volume=2|pages=900–926|doi=10.3390/v2040900}}</ref>
Although the RT structures from [[human]], [[murine]] and [[Bird|avian]] [[retroviruses]] display different subunits, the relative sizes, orientation and connection of the DNA polymerase and RNase H domains are strikingly similar. The RNase H domain occupies ~25% of the RT protein [[C-terminal]]. The DNA polymerase domain occupies ~55% of the RT protein N-terminal.<ref>{{cite journal|last=Beilhartz|first=GL|author2=Götte |title=HIV-1 ribonuclease H: structure, catalytic mechanism and inhibitors.|journal=Viruses|year=2010|volume=2|pages=900–926|doi=10.3390/v2040900}}</ref>
The RNase H domains of [[MMLV]] and [[HIV-1]] RT enzymes are structural very similar to the [[Escherichia coli]] and [[Bacillus halodurans]] RNases H as well as to human [[RNaseH1]].<ref>{{cite journal|last=Lim|first=David|author2=Steve Goff |title=Crystal structure of the moloney murine leukemia virus RNase H domain.|journal=Journal of Virology|month=September 2006|volume=80|issue=17|pages=8379–8389|doi=10.1128/jvi.00750-06}}</ref><ref>{{cite journal|last=KATAYANAGI|first=K|title=Three-dimensional structure of ribonuclease H from E. coli|journal=Nature|year=1990|volume=390|pages=306–309}}</ref><ref>{{cite journal|last=Yang|first=W|title=Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein.|journal=Science|year=1990|volume=249|issue=4975|pages=1398–405.|doi=10.1126/science.2169648}}</ref><ref>{{cite journal|last=Nowotny|first=Marcin|author2=Wei Yang |title=Crystal Structures of RNase H Bound to an RNA/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis.|journal=Cell|date=July 2005|volume=121|issue=7|pages=1005–1016|doi=10.1016/j.cell.2005.04.024}}</ref><ref>{{cite journal|last=Leo|first=Berit|author2=Birgitta Wöhrl |title=The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding|journal=Retrovirology|year=2012|volume=9|issue=73|pages=1742-4690}}</ref><ref>{{cite journal|last=Leo|first=Berit|author2=Birgitta Wöhrl |title=The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding|journal=Retrovirology|year=2012|volume=9|issue=73|pages=1742-4690}}</ref> In general, the folded structures of retroviral RNase H domains take the form of 5-stranded mixed [[beta sheets]] flanked by four [[alpha helices]] in an asymmetric distribution. A notable difference between the various RNase H proteins is the presence or absence of the C-helix (present in E. coli, MLV and human RNases H, absent in HIV-1, B. halodurans and ASLV RNases H), a positively charged alpha helix also referred to as the basic loop or protrusion.<ref>{{cite journal|last=Leo|first=Berit|author2=Birgitta Wöhrl |title=The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding|journal=Retrovirology|year=2012|volume=9|issue=73|pages=1742-4690}}</ref> It is believed to have a role in substrate binding.<ref>{{cite journal|last=Leo|first=Berit|author2=Birgitta Wöhrl |title=The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding|journal=Retrovirology|year=2012|volume=9|issue=73|pages=1742-4690}}</ref>
The RNase H domains of [[MMLV]] and [[HIV-1]] RT enzymes are structural very similar to the [[Escherichia coli]] and [[Bacillus halodurans]] RNases H as well as to human [[RNaseH1]].<ref>{{cite journal|last=Lim|first=David|author2=Steve Goff |title=Crystal structure of the moloney murine leukemia virus RNase H domain.|journal=Journal of Virology|month=September 2006|volume=80|issue=17|pages=8379–8389|doi=10.1128/jvi.00750-06}}</ref><ref>{{cite journal|last=KATAYANAGI|first=K|title=Three-dimensional structure of ribonuclease H from E. coli|journal=Nature|year=1990|volume=390|pages=306–309}}</ref><ref>{{cite journal|last=Yang|first=W|title=Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein.|journal=Science|year=1990|volume=249|issue=4975|pages=1398–405.|doi=10.1126/science.2169648}}</ref><ref>{{cite journal|last=Nowotny|first=Marcin|author2=Wei Yang |title=Crystal Structures of RNase H Bound to an RNA/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis.|journal=Cell|date=July 2005|volume=121|issue=7|pages=1005–1016|doi=10.1016/j.cell.2005.04.024}}</ref><ref>{{cite journal|last=Leo|first=Berit|author2=Birgitta Wöhrl |title=The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding|journal=Retrovirology|year=2012|volume=9|issue=73|pages=1742-4690|doi=10.1186/1742-4690-9-73}}</ref><ref>{{cite journal|last=Leo|first=Berit|author2=Birgitta Wöhrl |title=The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding|journal=Retrovirology|year=2012|volume=9|issue=73|pages=1742-4690|doi=10.1186/1742-4690-9-73}}</ref> In general, the folded structures of retroviral RNase H domains take the form of 5-stranded mixed [[beta sheets]] flanked by four [[alpha helices]] in an asymmetric distribution. A notable difference between the various RNase H proteins is the presence or absence of the C-helix (present in E. coli, MLV and human RNases H, absent in HIV-1, B. halodurans and ASLV RNases H), a positively charged alpha helix also referred to as the basic loop or protrusion.<ref>{{cite journal|last=Leo|first=Berit|author2=Birgitta Wöhrl |title=The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding|journal=Retrovirology|year=2012|volume=9|issue=73|pages=1742-4690|doi=10.1186/1742-4690-9-73}}</ref> It is believed to have a role in substrate binding.<ref>{{cite journal|last=Leo|first=Berit|author2=Birgitta Wöhrl |title=The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding|journal=Retrovirology|year=2012|volume=9|issue=73|pages=1742-4690|doi=10.1186/1742-4690-9-73}}</ref>
[[File:Retroviral genomes.png|right|x150px|Retroviral genomes.png|thumb|Typical retroviral genomic organization: The RNase H domain (RH - shown in black) is encoded as part of the [[reverse transcriptase]] (RT) [[gene]]. RT together with [[protease]] (PR) and [[integrase]] (IN) are translated as the [[Gag polyprotein]]. UTR = [[Untranslated region]]; LTR = [[Long terminal repeat]]]]
[[File:Retroviral genomes.png|right|x150px|Retroviral genomes.png|thumb|Typical retroviral genomic organization: The RNase H domain (RH - shown in black) is encoded as part of the [[reverse transcriptase]] (RT) [[gene]]. RT together with [[protease]] (PR) and [[integrase]] (IN) are translated as the [[Gag polyprotein]]. UTR = [[Untranslated region]]; LTR = [[Long terminal repeat]]]]


==Function==
==Function==


During [[reverse transcription]] of the viral genomic RNA into cDNA, an RNA/DNA hybrid is created. The RNA strand is then hydrolyzed by the RNase H domain to enable synthesis of the second DNA strand by the DNA polymerase function of the RT enzyme.<ref>{{cite journal|last=Beilhartz|first=GL|author2=Götte |title=HIV-1 ribonuclease H: structure, catalytic mechanism and inhibitors.|journal=Viruses|year=2010|volume=2|pages=900–926|doi=10.3390/v2040900}}</ref> In addition, retroviral [[virions]] package a single [[tRNA]] molecule that they use as a [[primer]] during reverse transcription of the viral genomic RNA.<ref>{{cite journal|last=Fu|first=Tie-Bo|author2=John taylor |title=When retroviral reverse transcriptases reach the end of their RNA templates|journal=Journal of Virology|date=27 March 1992|volume=66|issue=7|pages=4271–4278}}</ref> The retroviral RNase H is needed to digest the tRNA molecule when it is no longer needed. These processes happen in a Mg2+ dependent fashion.<ref>{{cite journal|last=Taylor|first=JM|title=An analysis of the role of tRNA species as primers for the transcription into DNA of RNA tumor virus genomes.|journal=Biochimica et Biophysica Acta|date=March 21, 1977|volume=473|issue=1|pages=:57–71|doi=10.1016/0304-419x(77)90007-5}}</ref><ref>{{cite journal|last=Talele|first=Tanaji|author2=Virendra Pandey |title=Influence of the RNase H domain of retroviral reverse transcriptases on the metal specificity and substrate selection of their polymerase domains|journal=Virology Journal|date=8 October 2009|volume=6|issue=159|pages=1743-4226}}</ref>
During [[reverse transcription]] of the viral genomic RNA into cDNA, an RNA/DNA hybrid is created. The RNA strand is then hydrolyzed by the RNase H domain to enable synthesis of the second DNA strand by the DNA polymerase function of the RT enzyme.<ref>{{cite journal|last=Beilhartz|first=GL|author2=Götte |title=HIV-1 ribonuclease H: structure, catalytic mechanism and inhibitors.|journal=Viruses|year=2010|volume=2|pages=900–926|doi=10.3390/v2040900}}</ref> In addition, retroviral [[virions]] package a single [[tRNA]] molecule that they use as a [[primer]] during reverse transcription of the viral genomic RNA.<ref>{{cite journal|last=Fu|first=Tie-Bo|author2=John taylor |title=When retroviral reverse transcriptases reach the end of their RNA templates|journal=Journal of Virology|date=27 March 1992|volume=66|issue=7|pages=4271–4278}}</ref> The retroviral RNase H is needed to digest the tRNA molecule when it is no longer needed. These processes happen in a Mg2+ dependent fashion.<ref>{{cite journal|last=Taylor|first=JM|title=An analysis of the role of tRNA species as primers for the transcription into DNA of RNA tumor virus genomes.|journal=Biochimica et Biophysica Acta|date=March 21, 1977|volume=473|issue=1|pages=:57–71|doi=10.1016/0304-419x(77)90007-5}}</ref><ref>{{cite journal|last=Talele|first=Tanaji|author2=Virendra Pandey |title=Influence of the RNase H domain of retroviral reverse transcriptases on the metal specificity and substrate selection of their polymerase domains|journal=Virology Journal|date=8 October 2009|volume=6|issue=159|pages=1743-4226|doi=10.1186/1743-422x-6-159}}</ref>


Retroviral RNases H cleave their [[Substrate (biochemsitry)|substrates]] through 3 different modes:
Retroviral RNases H cleave their [[Substrate (biochemsitry)|substrates]] through 3 different modes:

Revision as of 08:15, 19 August 2014

Retroviral ribonuclease H
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EC no.3.1.26.13
CAS no.9050-76-4
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A 3D molecular reconstruction of an RNase H domain using Pymol

The retroviral ribonuclease H (retroviral RNase H) is a catalytic domain of the retroviral reverse transcriptase (RT) enzyme. The RT enzyme is used to generate complementary DNA (cDNA) from the retroviral RNA genome. This process is called reverse transcription. To complete this complex process, the retroviral RT enzymes need to adopt a multifunctional nature. They therefore possess 3 of the following biochemical activities: RNA-dependent DNA polymerase, ribonuclease H, and DNA-dependent DNA polymerase activities [1] ). Like all RNase H enzymes, the retroviral RNase H domain cleaves DNA/RNA duplexes and will not degrade DNA or unhybridized RNA.

Structure

Although the RT structures from human, murine and avian retroviruses display different subunits, the relative sizes, orientation and connection of the DNA polymerase and RNase H domains are strikingly similar. The RNase H domain occupies ~25% of the RT protein C-terminal. The DNA polymerase domain occupies ~55% of the RT protein N-terminal.[2] The RNase H domains of MMLV and HIV-1 RT enzymes are structural very similar to the Escherichia coli and Bacillus halodurans RNases H as well as to human RNaseH1.[3][4][5][6][7][8] In general, the folded structures of retroviral RNase H domains take the form of 5-stranded mixed beta sheets flanked by four alpha helices in an asymmetric distribution. A notable difference between the various RNase H proteins is the presence or absence of the C-helix (present in E. coli, MLV and human RNases H, absent in HIV-1, B. halodurans and ASLV RNases H), a positively charged alpha helix also referred to as the basic loop or protrusion.[9] It is believed to have a role in substrate binding.[10]

Typical retroviral genomic organization: The RNase H domain (RH - shown in black) is encoded as part of the reverse transcriptase (RT) gene. RT together with protease (PR) and integrase (IN) are translated as the Gag polyprotein. UTR = Untranslated region; LTR = Long terminal repeat

Function

During reverse transcription of the viral genomic RNA into cDNA, an RNA/DNA hybrid is created. The RNA strand is then hydrolyzed by the RNase H domain to enable synthesis of the second DNA strand by the DNA polymerase function of the RT enzyme.[11] In addition, retroviral virions package a single tRNA molecule that they use as a primer during reverse transcription of the viral genomic RNA.[12] The retroviral RNase H is needed to digest the tRNA molecule when it is no longer needed. These processes happen in a Mg2+ dependent fashion.[13][14]

Retroviral RNases H cleave their substrates through 3 different modes:

  1. sequence-specific internal cleavage of RNA [1-4]. Human immunodeficiency virus type 1 and Moloney murine leukemia virus enzymes prefer to cleave the RNA strand one nucleotide away from the RNA-DNA junction.
  2. RNA 5'-end directed cleavage 13-19 nucleotides from the RNA end.
  3. DNA 3'-end directed cleavage 15-20 nucleotides away from the primer terminus.

The two end-directed modes are unique to the retroviral RNases H because of a number of effects of the associated polymerase domain of retroviral RT.[15] In the more universal internal cleavage mode, the RNases H behave as typical endonucleases and cleave the RNA along the length of a DNA / RNA hybrid substrate in the absence of any ‘end’ effects.[16][17][18][19]

References

  1. ^ Worthington, Von (1993). Worthington Enzyme Manual. Worthington. p. 280.
  2. ^ Beilhartz, GL; Götte (2010). "HIV-1 ribonuclease H: structure, catalytic mechanism and inhibitors". Viruses. 2: 900–926. doi:10.3390/v2040900.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  3. ^ Lim, David; Steve Goff. "Crystal structure of the moloney murine leukemia virus RNase H domain". Journal of Virology. 80 (17): 8379–8389. doi:10.1128/jvi.00750-06. {{cite journal}}: Unknown parameter |month= ignored (help)
  4. ^ KATAYANAGI, K (1990). "Three-dimensional structure of ribonuclease H from E. coli". Nature. 390: 306–309.
  5. ^ Yang, W (1990). "Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein". Science. 249 (4975): 1398–405. doi:10.1126/science.2169648.
  6. ^ Nowotny, Marcin; Wei Yang (July 2005). "Crystal Structures of RNase H Bound to an RNA/DNA Hybrid: Substrate Specificity and Metal-Dependent Catalysis". Cell. 121 (7): 1005–1016. doi:10.1016/j.cell.2005.04.024.
  7. ^ Leo, Berit; Birgitta Wöhrl (2012). "The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding". Retrovirology. 9 (73): 1742–4690. doi:10.1186/1742-4690-9-73.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  8. ^ Leo, Berit; Birgitta Wöhrl (2012). "The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding". Retrovirology. 9 (73): 1742–4690. doi:10.1186/1742-4690-9-73.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  9. ^ Leo, Berit; Birgitta Wöhrl (2012). "The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding". Retrovirology. 9 (73): 1742–4690. doi:10.1186/1742-4690-9-73.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  10. ^ Leo, Berit; Birgitta Wöhrl (2012). "The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding". Retrovirology. 9 (73): 1742–4690. doi:10.1186/1742-4690-9-73.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  11. ^ Beilhartz, GL; Götte (2010). "HIV-1 ribonuclease H: structure, catalytic mechanism and inhibitors". Viruses. 2: 900–926. doi:10.3390/v2040900.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  12. ^ Fu, Tie-Bo; John taylor (27 March 1992). "When retroviral reverse transcriptases reach the end of their RNA templates". Journal of Virology. 66 (7): 4271–4278.
  13. ^ Taylor, JM (March 21, 1977). "An analysis of the role of tRNA species as primers for the transcription into DNA of RNA tumor virus genomes". Biochimica et Biophysica Acta. 473 (1): :57–71. doi:10.1016/0304-419x(77)90007-5.
  14. ^ Talele, Tanaji; Virendra Pandey (8 October 2009). "Influence of the RNase H domain of retroviral reverse transcriptases on the metal specificity and substrate selection of their polymerase domains". Virology Journal. 6 (159): 1743–4226. doi:10.1186/1743-422x-6-159.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  15. ^ Lim, David; Steve Goff. "Crystal structure of the moloney murine leukemia virus RNase H domain". Journal of Virology. 80 (17): 8379–8389. doi:10.1128/jvi.00750-06. {{cite journal}}: Unknown parameter |month= ignored (help)
  16. ^ Schultz, S.J., Zhang, M. and Champoux, J.J. (2004). "Recognition of internal cleavage sites by retroviral RNases H". J. Mol. Biol. 344 (3): 635–652. doi:10.1016/j.jmb.2004.09.081. PMID 15533434.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  17. ^ Krug, M.S. and Berger, S.L. (1989). "Ribonuclease H activities associated with viral reverse transcriptases are endonucleases". Proc. Natl. Acad. Sci. USA. 86 (10): 3539–3543. Bibcode:1989PNAS...86.3539K. doi:10.1073/pnas.86.10.3539. PMC 287173. PMID 2471188.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  18. ^ Champoux, J.J. and Schultz, S.J. (2009). "Ribonuclease H: properties, substrate specificity and roles in retroviral reverse transcription". FEBS J. 276 (6): 1506–1516. doi:10.1111/j.1742-4658.2009.06909.x. PMC 2742777. PMID 19228195.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  19. ^ Schultz, S.J. and Champoux, J.J. (2008). "RNase H activity: structure, specificity, and function in reverse transcription". Virus Res. 134 (1–2): 86–103. doi:10.1016/j.virusres.2007.12.007. PMC 2464458. PMID 18261820.{{cite journal}}: CS1 maint: multiple names: authors list (link)
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