Sequence, Distance, and Sequence, Distance, and Accessibility are Determinants of 5' Accessibility are Determinants of 5'

End-DirectedEnd-DirectedCleavages by Retroviral RNaseCleavages by Retroviral RNase

Sharon J. Schultz, Sharon J. Schultz, et al. (2005). et al. (2005). J. Biol. Chem.J. Biol. Chem.M510504200M510504200

生科四甲生科四甲報告學生報告學生 :: 李殿強李殿強

Introduction:Introduction:• Retroviral replicationRetroviral replication

RNA virus:RNA virus:

Minus-strand DNA( - )Minus-strand DNA( - )

Plus-strand DNA(+)Plus-strand DNA(+)

專有名詞介紹專有名詞介紹 ::

• Reverse transcriptaseReverse transcriptase

• Moloney murine leukemia virus(M-Moloney murine leukemia virus(M-

MuLV)MuLV)

• The reverse transcriptase of HIV-1The reverse transcriptase of HIV-1

• The reverse transcriptase of M-MuLVThe reverse transcriptase of M-MuLV

• polypurine tract (PPT)polypurine tract (PPT)

RNase H contributes to reverseRNase H contributes to reversetranscription in three distinct transcription in three distinct ways:ways:

• First, RNase H carries out degradation of First, RNase H carries out degradation of the RNA genome both during and after the RNA genome both during and after minus-strand DNA synthesis to facilitate minus-strand DNA synthesis to facilitate plus-strand DNA synthesis.plus-strand DNA synthesis.

• Second, RNase H creates the polypurine Second, RNase H creates the polypurine tract (PPT) primer from the viral genome.tract (PPT) primer from the viral genome.

• third, RNase H removes the PPT and tRNA third, RNase H removes the PPT and tRNA primers used to prime plus-strand and primers used to prime plus-strand and minus-stand DNA synthesis.minus-stand DNA synthesis.

Asking three fundamental Asking three fundamental questions concerning retroviral questions concerning retroviral RNases H?RNases H?

• First, does sequence influence 5' First, does sequence influence 5' end- directed cleavages?end- directed cleavages?

• Second, what distances from the RNA Second, what distances from the RNA 5' end are acceptable for 5'end-5' end are acceptable for 5'end-directed cleavage sites?directed cleavage sites?

• Third, how large of a gap is required Third, how large of a gap is required between the 3' terminus of an between the 3' terminus of an upstream RNA and the 5' end of a upstream RNA and the 5' end of a downstream RNA?downstream RNA?

EXPERIMENT:EXPERIMENT:Enzymes and reagents–Enzymes and reagents–

• HIV-1 reverse transcriptase HIV-1 reverse transcriptase

• M-MuLV reverse transcriptaseM-MuLV reverse transcriptase

• T7 DNA polymeraseT7 DNA polymerase

• T4 polynucleotide kinase, T4 DNA T4 polynucleotide kinase, T4 DNA ligase, T4 DNA polymeraseligase, T4 DNA polymerase

• restriction enzymesrestriction enzymes

• Gel-purified in denaturing Gel-purified in denaturing polyacrylamide gels(polyacrylamide gels( 跑跑 RNARNA 用用 ))

Preparation ofPreparation of RNAs:RNAs:pGEM- Vector SystemspGEM- Vector SystemsMd1 – Md10, PPT20, PPT62, and NPPTMd1 – Md10, PPT20, PPT62, and NPPT

Preparation of hybrid substratesPreparation of hybrid substrates::RNAs were annealed to the RNAs were annealed to the appropriate template DNAsappropriate template DNAs

T7 RNA polymeraseT7 RNA polymerase

Template-dependentTemplate-dependent

RNAs:RNAs:

Result:Result:HIV-1 5´ end-labeling HIV-1 5´ end-labeling

I +27/+28I +27/+28

H H +22/+23+22/+23

G G +19/+20+19/+20

F +16/+17F +16/+17

E +13/+14E +13/+14

HIV-1 3´ end-labelingHIV-1 3´ end-labeling

M-MuLV 5´ end-labeling M-MuLV 5´ end-labeling

M-MuLV 3´ end-labelingM-MuLV 3´ end-labeling

Distance:Distance:

HIV-1HIV-1

M-MuLVM-MuLV

Prefer Base:Prefer Base:

EXPERIMENT:EXPERIMENT:GAP SIZEGAP SIZE: : M-MuLVM-MuLV

HIV-1HIV-1

ConclusionConclusion::

1.1.An alignment of sequences reveals that An alignment of sequences reveals that both enzymes strongly prefer both enzymes strongly prefer A or U at A or U at the +1 positionthe +1 position and and C or G at the –2 C or G at the –2 positionposition, and additionally for HIV-1, A is , and additionally for HIV-1, A is disfavored at the –4 position.disfavored at the –4 position.

2.2.For both enzymes, 5' end-directed For both enzymes, 5' end-directed cleavages occurred when sites were cleavages occurred when sites were positioned between the 13th and 20th positioned between the 13th and 20th nucleotides from the RNA 5' end, a nucleotides from the RNA 5' end, a distance termed the distance termed the cleavage windowcleavage window..

3.3.The importance of accessibility to the RNA The importance of accessibility to the RNA 5' end, it was found that the 5' end, it was found that the extent of 5' end-directed cleavages extent of 5' end-directed cleavages observed in substrates was most observed in substrates was most comparable to substrates with a gap of comparable to substrates with a gap of 2 or 2 or 33 bases between the upstream and bases between the upstream and downstream RNAs.downstream RNAs.

The End