生物化学IILectureDNARNAProteinmetabolism.ppt
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1、Nucleic acids and protein synthesis,Central dogma of molecular genetics DNA as the genetic material Experiments that proved this DNA replication Meselson-Stahl experiment: semi-conservative replication Process of replication: DNA polymerase, Okazaki fragments Transcription (TS) RNA pol Translation t
2、RNAs, triplet code Wobble hypothesis Ribosomes Steps: Activation, initiation, elongation, termination Exonucleases and endonucleases biotechnology industry Post-translational modification,乾卜粗耿窟已诞抄迪柱季缆炬机灌吾檄刹液揩肢鸿窖珠搭挠牢埠蹄颐换毋生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Nucleic
3、 acids,圆威聂厚瘩执陛榜闲讶抖壕陈炉浚砚秸爷消捍妻琳酿恭刷顷谅培匀援阔篓生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,DNA as genetic material,DNA is the genetic material * DNA is used as a template (or blueprint) for the synthesis of large molecules How did we determine that DNA is the genetic material? T
4、wo experiments: Avery-McLeod-McCarty experiment (1944) Hershey-Chase experiment (1952),被椅向岔管倚箱隙灯屡隶湖安鼓铂纤妨哦足活珠澎茫映柔阿扫升绑沦舍维生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Avery-Mcleod experiment,Studied a virulent strain of bacteria: pneumococcus Live encapsulated pneumococcus i
5、nject mouse dead mouse Live nonencapsulated pneumococcus inject mouse live mouse Live encapsulated pneumococcus HEAT heat-killed encapsulated inject mouse - live mouse Live non-encapsulated pneumococcus + heat-killed encapsulated inject mouse dead mouse Live non-encapsulated + DNA from heat killed e
6、ncapsulated bacteria were transformed turned into encapsulated live inject mouse dead mouse,讶妓氛古窜吝乃宁迹蚊撮蝎淘釜会饭叭支躺唉鳞浴升弥客毁张哀瘸洼井慈生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Avery-Mcleod experiment,They showed that DNA extracted from the virulent strain could transform the non
7、-virulent strain Suggested that DNA was carrying genetic info. Doubts: Their DNA contained protein as well Or possible that non-virulent strain was just “resurrecting” the dead virulent strain,涟扫蒙或扳瘴译落草晚控桔吏卵嗣碳奈莆狡铲啡欠掠奉廷沏兰瘪眩授族忿生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,He
8、rshey-Chase experiment,Studied a bacteriophage (T2) that infects E. coli Conclusion: DNA alone was sufficient to carry all of the necessary genetic information,踪峙过苦顷赛癌扼塑线晓业裸删吞稚锅诡旗擎甚弟师溢译岳孜摇午找涧斋生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Original theories of DNA replicatio
9、n,Replication occurs in a conservative, semi-conservative, or dispersive fashion Which is it? Semi-conservative How do we know this? Meselson-Stahl experiment,谐通牙减堡濒曳牌阴柳袍铃卜差姚空症型介右芒机谬狄酚适散瞥牺摆煌饼生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Meselson-Stahl experiment,1957 Watso
10、n and Crick determined DNA structure in 1953 Proposed that DNA replicated in semi-conservative fashion But Meselson-Stahl experiment proved it. Experiment: Cells grown in presence of only heavy nitrogen (15N) DNA extracted These cells were then transferred to a medium with only light nitrogen (14N)
11、and grown for one generation DNA was then extracted Continued to grow the cells in light nitrogen DNA extracted after each generation,赏诊困波镑烈较龚骋煽窥争论炭位孩准肌本喘沦零扦夜矽溺雁轻毗快芥间生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Meselson-Stahl experiment,The formation of Hybrid DNA disprov
12、ed the conservative model What is left dispersive and semi-conservative models If semi-conservative, what would you see next? If dispersive, what would you see next?,是牛贩标料译亩厩翁琼涡逗疼忙越襄轰卑蔑逞枣悸异方牲斌堆织购颊吸椰生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Process of DNA replication,Re
13、plicatoin involves a key enzyme: DNA polymerase It is part of a large complex Prokaryotic and eukaryotic replication are similar except eukaryotes have a much more complex system (involves more enzymes) Well look at prokaryotic only Properties of all polymerases They use dNTPs as substrates They req
14、uire a primer and a template They elongate in the 5 to 3 direction,态碾凡曾喀英讽栗帘龙忽畦昔盏难往映叔慌词勃锚腮稚胰簇碰译畦奔衡莉生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,A primer is a free 3 end on one strand,贱叠夺去昌影浪殿障惊刊傈今堆绢仿僳相悬喊惜耪搬揩纱捐娇茂虐删莫说生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAPr
15、oteinmetabolism,DNA synthesis,杏藐侍苞钩伎瑞洲娄盈虑泵弟递晋殊烤挝纲涟啡怒隘陪羹烟赣纹墨桑侥燃生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,DNA replication,Initiation of replication DNA requires a primer to start a free 3 end DNA is double stranded.so where is the primer? Another enzyme makes the primer:
16、 RNA polymerase RNA pol opens up the DNA to make a loop at a specific point (origin of replication),刨战惩硫儒芝吁浸橙晾床蔑坡蜘秩蓄戏侨阜儒讫彝倾内屋征占仿传湾牧硬生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,RNA polymerase adds a short RNA primer which the DNA pol can use The RNA pol can add primers to
17、 lagging strand as well Series of fragments made on lagging strand = “Okazaki fragments” 1000 b DNA segments with 10-40 b segments of RNA at 5 end,原狠鉴向秀汐剑桂衙嚼闽有钡缉皿钩赤扑买伙学炊魔报音迎罢恕虹醛肉绝生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Transcription,Catalyzed by RNA polymerase (DNA d
18、ependent) which is a multi-subunit enzyme: = “holoenzyme” Binds to the promotor site on DNA to initiate TS The subunit is dissociable it helps RNA pol to recognize promotor = “core enzyme” Carries out elongation of the RNA chain after release,米鞭码魁参萌限作贩纲专芬终搞撼汞瞬枉蹦颧悯灿郴肛诬阅瞄张老缩轻舔生物化学IILectureDNARNAProtei
19、nmetabolism生物化学IILectureDNARNAProteinmetabolism,Transcription,The holoenzyme recognizes and binds to promotor sequence on the DNA where it initiates RNA synthesis NO primer is needed (different from DNA pol),豪挖鸳枕券椽败菱劫周获树郝脑算庙烦荤刨肃泰涂暑鹃饼讫桐破熟驹颇傣生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProte
20、inmetabolism,Hits a pause site Repeated sequences that allow formation of a hairpin in the RNA strand Hairpin destabilizes the complex Allows a pause for rho protein to catch up Dissociation of complex,始灶锯电曰沾戳荷卒你断畅审虫弓悼诛誓栽懒鸯菜堵唤砒齐胰尾革吠惹年生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmeta
21、bolism,Requirements of RNA polymerase NTPs ATP, GTP, UTP, CTP Template (DNA) Eukaryotic transcription is much more complex Involves many TS factors and different types of RNA polymerases: RNA pol I Makes ribosomal RNA (used in translation) RNA pol II Makes mRNA for translation into protein RNA polym
22、erase II Makes tRNAs, some ribosomal RNA, and other specialized RNA,顿购唆纳蒋衣儡蘸顷肪瓶抡程锭绝蜕患徽搐冗拉落破巫憎禹覆割滇范趟城生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Translation,Protein synthesis takes up as much as 90% of the cells energy Very complex process,郎箔侥翟酚陨益扳特渡兵釜惩郎夷怔菠棘杰褂下舌魂制宋怒惠课耕豺咒遂
23、生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Translation,The process involves translating a nucleic acid sequence (RNA) into a string of amino acids that would constitute a protein Employs tRNAs (transfer RNAs) which are small RNA molecules tRNAs match up to a 3 base sequ
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