Siu Chun Hung - Academia.edu (original) (raw)

Papers by Siu Chun Hung

Journal of Bacteriology, 1995

The rate of transcription elongation in Escherichia coli was reduced when cells were depleted of ... more The rate of transcription elongation in Escherichia coli was reduced when cells were depleted of NusG. In a purified in vitro system, NusG accelerated the transcription elongation rate. The stimulation of the rate of transcription elongation by NusG appears to result from the suppression of specific transcription pause sites.

Proceedings of the National Academy of Sciences, 2001

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP , enables ... more EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP , enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1–89 and 323–386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379–641), which includes the nuclear localization signal and DNA-binding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP -containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1–378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1–90 or histone H1–2 could substitute for EB...

Proceedings of the National Academy of Sciences, 1998

The Epstein–Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively... more The Epstein–Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively aggregated pseudo-tumor necrosis factor receptor (TNFR) that activates transcription factor NF-κB through two sites in its C-terminal cytoplasmic domain. One site is similar to activated TNFRII in associating with TNFR-associated factors TRAF1 and TRAF2, and the second site is similar to TNFRI in associating with the TNFRI death domain interacting protein TRADD. TNFRI has been recently shown to activate NF-κB through association with TRADD, RIP, and TRAF2; activation of the NF-κB-inducing kinase (NIK); activation of the IκBα kinases (IKKα and IKKβ); and phosphorylation of IκBα. IκBα phosphorylation on Ser-32 and Ser-36 is followed by its degradation and NF-κB activation. In this report, we show that NF-κB activation by LMP1 or by each of its effector sites is mediated by a pathway that includes NIK, IKKα, and IKKβ. Dominant negative mutants of NIK, IKKα, or IKKβ substantially inhibited ...

Proceedings of the National Academy of Sciences, 1995

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression... more The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and ...

Proceedings of the National Academy of Sciences, 2005

The lymphoma-inducing potential of Ig heavy-chain enhancer- and promoter-regulated Epstein-Barr v... more The lymphoma-inducing potential of Ig heavy-chain enhancer- and promoter-regulated Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was evaluated in three transgenic FVB mouse lineages. EBNA1 was expressed at a higher level in transgenic B220(+) splenocytes than in EBV-infected lymphoblastoid cell lines. EBNA1 was also expressed in B220(-) transgenic splenocytes and thymocytes. Before killing and assessments at 18-26 months, EBNA1-transgenic mice did not differ from control mice in mortality. At 18-26 months EBNA1-transgenic mice did not differ from littermate control in ultimate body weight, in spleen size or weight, in lymph node, kidney, liver, or spleen histology, in splenocyte fractions positive for cluster of differentiation (CD)3ε, CD4, CD8, CD62L, B220, CD5, IgM, IgD, MHC class II, CD11b, or CD25, or in serum IgM, IgG, or total Ig levels. Lymphomas were not found in spleens or other organs of 18- to 26-month-old EBNA1-transgenic ( n = 86) or control ( n = 45) FVB mice. EBN...

Proceedings of the National Academy of Sciences, 2001

By binding to a cis-acting element ( oriP ) in the Epstein–Barr virus (EBV) genome, EBV nuclear a... more By binding to a cis-acting element ( oriP ) in the Epstein–Barr virus (EBV) genome, EBV nuclear antigen 1 (EBNA1) enables persistence and enhances transcription from EBV episomes. To investigate whether EBNA1 also directly affects cell gene transcription, we conditionally expressed a Flag-tagged dominant negative EBNA1 (FDNE) in an EBV immortalized lymphoblastoid cell line, in which the EBV genome is integrated into cell DNA. FDNE induction inhibited expression from an EBNA1-dependent oriP reporter plasmid by more than 90% in these cells but did not affect expression from integrated EBV or oriP reporter DNA. FDNE induction also did not alter expression of more than 1,800 cellular mRNAs. Lymphoblastoid cell line growth under a variety of conditions was unaffected by FDNE induction. Although Gal4-VP16 and EBNA1 strongly activated and coactivated a Gal4-VP16- and oriP-dependent promoter that was on an episome, only Gal4-VP16 activated the promoter when it was integrated into chromosoma...

Molecular Microbiology, 1999

The Escherichia coli nusG gene product is required for transcription termination by phage HK022 N... more The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the nutR site in vivo. We show that it is also essential for Nun termination at nutL. Three recessive missense nusG mutations have been isolated that inhibit termination by Nun at nutR. The mutations are ineffective in a pL nutL fusion, even when nutR replaces nutL. The mutant strains support growth, indicating that N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both nutR and nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.

Molecular Microbiology, 1994

Escherichia coliDnaK, DnaJ and GrpE are required for renaturation of heat-inactivated /. CI857 re... more Escherichia coliDnaK, DnaJ and GrpE are required for renaturation of heat-inactivated /. CI857 repressor (Gaitanaris et ai.., 1990). Here we demonstrate that in addition to the above three proteins, GroEL and GroES are necessary for the CI857 repressor to acquire full activity at the permissive temperature. Although full-length soluble repressor is present at normal amounts, the protein has reduced specific activity and migrates abnormally on native gels. To determine where the different chaperones act in protein folding, we identified their cellular locations. DnaK and DnaJ are associated with nascent polypeptide chains in translating ribosomes. In contrast, GroEL, although it is transiently associated with newiy synthesized proteins, is absent from the ribosomes. This suggests that DnaK and DnaJ ptay an early role in protein maturation, whereas GroEL acts at a later stage.

Journal of Molecular Biology, 1995

Phage HK022 Nun protein excludes phage l by terminating transcription 1 Department of Biochemistr... more Phage HK022 Nun protein excludes phage l by terminating transcription 1 Department of Biochemistry near the l nut sites. We have established a purified in vitro system that and Molecular Biophysics reproduces the in vivo sequence and factor requirements of Nun. Nun arrests 2 Institute of Cancer Research transcription by E. coli RNA polymerase at or near elongation pause sites College of Physicians and Surgeons, Columbia distal to the nut sites. The boxB sequence of nut is required for optimal Nun University, New York activity; boxA plays a lesser role. The efficiency of transcription arrest is strongly enhanced by the four E. coli Nus factors. The factors increase the NY 10032, U.S.A. specific activity of Nun, and allow it to act at higher ribonucleoside triphosphate concentrations. A wild-type boxA is required for stimulation by Nus factors. Nun and the l N antitermination protein compete for their opposing reactions. This competition may be at the level of binding of boxB RNA.

Genes & Development, 1997

Bacteriophage HK022 Nun protein blocks transcription elongation byEscherichia coli RNA polymerase... more Bacteriophage HK022 Nun protein blocks transcription elongation byEscherichia coli RNA polymerase in vitro without dissociating the transcription complex. Nun is active on complexes located at any template site tested. Ultimately, only the 3′-OH terminal nucleotide of the nascent transcript in an arrested complex can turn over; it is removed by pyrophosphate and restored with NTPs. This suggests that Nun inhibits the translocation of RNA polymerase without abolishing its catalytic activities. Unlike spontaneously arrested complexes, Nun-arrested complexes cannot be reactivated by transcription factor GreB. The various complexes show distinct patterns of nucleotide incorporation and pyrophosphorolysis before or after treatment with Nun, suggesting that the configuration of RNAP, transcript, and template DNA is different in each complex.

Journal of Bacteriology, 1995

The rate of transcription elongation in Escherichia coli was reduced when cells were depleted of ... more The rate of transcription elongation in Escherichia coli was reduced when cells were depleted of NusG. In a purified in vitro system, NusG accelerated the transcription elongation rate. The stimulation of the rate of transcription elongation by NusG appears to result from the suppression of specific transcription pause sites.

Proceedings of the National Academy of Sciences, 2001

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP , enables ... more EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP , enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1–89 and 323–386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379–641), which includes the nuclear localization signal and DNA-binding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP -containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1–378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1–90 or histone H1–2 could substitute for EB...

Proceedings of the National Academy of Sciences, 1998

The Epstein–Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively... more The Epstein–Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively aggregated pseudo-tumor necrosis factor receptor (TNFR) that activates transcription factor NF-κB through two sites in its C-terminal cytoplasmic domain. One site is similar to activated TNFRII in associating with TNFR-associated factors TRAF1 and TRAF2, and the second site is similar to TNFRI in associating with the TNFRI death domain interacting protein TRADD. TNFRI has been recently shown to activate NF-κB through association with TRADD, RIP, and TRAF2; activation of the NF-κB-inducing kinase (NIK); activation of the IκBα kinases (IKKα and IKKβ); and phosphorylation of IκBα. IκBα phosphorylation on Ser-32 and Ser-36 is followed by its degradation and NF-κB activation. In this report, we show that NF-κB activation by LMP1 or by each of its effector sites is mediated by a pathway that includes NIK, IKKα, and IKKβ. Dominant negative mutants of NIK, IKKα, or IKKβ substantially inhibited ...

Proceedings of the National Academy of Sciences, 1995

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression... more The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and ...

Proceedings of the National Academy of Sciences, 2005

The lymphoma-inducing potential of Ig heavy-chain enhancer- and promoter-regulated Epstein-Barr v... more The lymphoma-inducing potential of Ig heavy-chain enhancer- and promoter-regulated Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was evaluated in three transgenic FVB mouse lineages. EBNA1 was expressed at a higher level in transgenic B220(+) splenocytes than in EBV-infected lymphoblastoid cell lines. EBNA1 was also expressed in B220(-) transgenic splenocytes and thymocytes. Before killing and assessments at 18-26 months, EBNA1-transgenic mice did not differ from control mice in mortality. At 18-26 months EBNA1-transgenic mice did not differ from littermate control in ultimate body weight, in spleen size or weight, in lymph node, kidney, liver, or spleen histology, in splenocyte fractions positive for cluster of differentiation (CD)3ε, CD4, CD8, CD62L, B220, CD5, IgM, IgD, MHC class II, CD11b, or CD25, or in serum IgM, IgG, or total Ig levels. Lymphomas were not found in spleens or other organs of 18- to 26-month-old EBNA1-transgenic ( n = 86) or control ( n = 45) FVB mice. EBN...

Proceedings of the National Academy of Sciences, 2001

By binding to a cis-acting element ( oriP ) in the Epstein–Barr virus (EBV) genome, EBV nuclear a... more By binding to a cis-acting element ( oriP ) in the Epstein–Barr virus (EBV) genome, EBV nuclear antigen 1 (EBNA1) enables persistence and enhances transcription from EBV episomes. To investigate whether EBNA1 also directly affects cell gene transcription, we conditionally expressed a Flag-tagged dominant negative EBNA1 (FDNE) in an EBV immortalized lymphoblastoid cell line, in which the EBV genome is integrated into cell DNA. FDNE induction inhibited expression from an EBNA1-dependent oriP reporter plasmid by more than 90% in these cells but did not affect expression from integrated EBV or oriP reporter DNA. FDNE induction also did not alter expression of more than 1,800 cellular mRNAs. Lymphoblastoid cell line growth under a variety of conditions was unaffected by FDNE induction. Although Gal4-VP16 and EBNA1 strongly activated and coactivated a Gal4-VP16- and oriP-dependent promoter that was on an episome, only Gal4-VP16 activated the promoter when it was integrated into chromosoma...

Molecular Microbiology, 1999

The Escherichia coli nusG gene product is required for transcription termination by phage HK022 N... more The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the nutR site in vivo. We show that it is also essential for Nun termination at nutL. Three recessive missense nusG mutations have been isolated that inhibit termination by Nun at nutR. The mutations are ineffective in a pL nutL fusion, even when nutR replaces nutL. The mutant strains support growth, indicating that N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both nutR and nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.

Molecular Microbiology, 1994

Escherichia coliDnaK, DnaJ and GrpE are required for renaturation of heat-inactivated /. CI857 re... more Escherichia coliDnaK, DnaJ and GrpE are required for renaturation of heat-inactivated /. CI857 repressor (Gaitanaris et ai.., 1990). Here we demonstrate that in addition to the above three proteins, GroEL and GroES are necessary for the CI857 repressor to acquire full activity at the permissive temperature. Although full-length soluble repressor is present at normal amounts, the protein has reduced specific activity and migrates abnormally on native gels. To determine where the different chaperones act in protein folding, we identified their cellular locations. DnaK and DnaJ are associated with nascent polypeptide chains in translating ribosomes. In contrast, GroEL, although it is transiently associated with newiy synthesized proteins, is absent from the ribosomes. This suggests that DnaK and DnaJ ptay an early role in protein maturation, whereas GroEL acts at a later stage.

Journal of Molecular Biology, 1995

Phage HK022 Nun protein excludes phage l by terminating transcription 1 Department of Biochemistr... more Phage HK022 Nun protein excludes phage l by terminating transcription 1 Department of Biochemistry near the l nut sites. We have established a purified in vitro system that and Molecular Biophysics reproduces the in vivo sequence and factor requirements of Nun. Nun arrests 2 Institute of Cancer Research transcription by E. coli RNA polymerase at or near elongation pause sites College of Physicians and Surgeons, Columbia distal to the nut sites. The boxB sequence of nut is required for optimal Nun University, New York activity; boxA plays a lesser role. The efficiency of transcription arrest is strongly enhanced by the four E. coli Nus factors. The factors increase the NY 10032, U.S.A. specific activity of Nun, and allow it to act at higher ribonucleoside triphosphate concentrations. A wild-type boxA is required for stimulation by Nus factors. Nun and the l N antitermination protein compete for their opposing reactions. This competition may be at the level of binding of boxB RNA.

Genes & Development, 1997

Bacteriophage HK022 Nun protein blocks transcription elongation byEscherichia coli RNA polymerase... more Bacteriophage HK022 Nun protein blocks transcription elongation byEscherichia coli RNA polymerase in vitro without dissociating the transcription complex. Nun is active on complexes located at any template site tested. Ultimately, only the 3′-OH terminal nucleotide of the nascent transcript in an arrested complex can turn over; it is removed by pyrophosphate and restored with NTPs. This suggests that Nun inhibits the translocation of RNA polymerase without abolishing its catalytic activities. Unlike spontaneously arrested complexes, Nun-arrested complexes cannot be reactivated by transcription factor GreB. The various complexes show distinct patterns of nucleotide incorporation and pyrophosphorolysis before or after treatment with Nun, suggesting that the configuration of RNAP, transcript, and template DNA is different in each complex.