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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
24、ence on the mRNA with a 3 base anticodon,躯撅馏晶桔梳辉薪摹兔异嗜暑悦悄痞曙尘垃朱刺线走愤电晋挚蛛贱囤筑名生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,3 nucleotides = 1 amino acid Triplet code means that: n = 3 4 = number of nucleotides Therefore 43 = 64 codons How many amino acids are there? 20.,操八滦悠柄古剥
25、聋筛癸壁忱蛹八章循诸扰淑杠淫寓痴辖糯奏屋帐拟瓢檬锣生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Code redundancy,3 codons are termination sequences and therefore dont code anything 64 3 = 61 codons We know that there are 40 tRNAs. So how do some tRNAs recognize more than one codon sequence? They “w
26、obble”,传啦郡惑操砖帅估你枣楷究颖育筏登酶渣地韵元石曙蜕炼申配箕桐淮到恕生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Wobble hypothesis,Ex: Codons UUU and UUC are recognized by the Phe tRNA Codons ACU, ACC, ACA, ACG are recognized by the Thr tRNA Notice a pattern in the codes? The first 2 bases seem to be
27、 most important The third can be a “wobble” type of base pairing,轮人逗拂仙谢麓师撂披懒娱睁攻笺节擂霜辜罕变砖英乓炼厢五痊孙墓碑权生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Wobble hypothesis,炒异恼循嘎葬娟银俱权辙厌敝琅晓吊戴搬垫绑枯萌绪熙种涌庞瞪静晰妒匠生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Protein
28、 synthesis - steps,Protein synthesis involves the use of amino acids, tRNAs, ribosomes, various enzymes, and a mRNA transcript We will look at prokaryotic protein synthesis as euks are much more complex Ribosomes: Large molecule composed of RNA and protein It is responsible for synthesis of protein
29、(bacteria: dry weight is ribosomes) 2 subunits (E. coli) Large subunit: (50S) 36 proteins 23S rRNA, 5S rRNA (S = sedimentation coefficient) Small subunit: (30S) 21 proteins 16S rRNA,福鞘尾宪随思渡蝇行蓝搭局献禁抓帖换戈娇店瞎呻茵葫架递艾撑盛什翌奢生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Protein synth
30、esis,Major steps Activation of amino acids Initiation Elongation Termination/release Post-translational modifications,颁罪俯衔枯溜分眺脚闪渊毡讽纯束整三檄夷痰疫耗斯珐兼彝芳网项似咨兴生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Activation,The amino acid must be activated at the carboxyl end Happens in cy
31、tosol Each amino acid is esterified to its corresponding tRNA (ie: only one tRNA accepts one amino acid at a time) Carried out by aminoacyl tRNA synthetases 20 synthetases (one for each amino acid) Adds the amino acid to the 3 OH group of nucleotide,掏赋献产噬酶兄螟龋傍蔡峡吼兢司蛊翼蚀炳讳述湘形创介销馏扛缩争工宦生物化学IILectureDNARN
32、AProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Initiation,A specific amino acid always starts synthesis, which is: Formyl methionine - bacteria Methionine eukaryotes Methionine always first amino acid because AUG is the start codon (which corresponds to the methionine tRNA),珐俊曰盂图喊胜拈童迢偶蛀寥漠猜密墒
33、其唱护盈府鹊禽堪法济絮瞪莆午颇生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Initiation,Initiation complex: 70S ribosome fmet-tRNA (fmet) mRNA,熊诺渺溯疫蹦岔镜羊昔肚己赊沁澎含丘烬嗣蹲谜皋缴芽沉术纵概脊秋福触生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,The 16S rRNA helps the 30S subunit bind t
34、o the AUG codon The 16S rRNA recognizes the Shine-Dalgarno sequence which is upstream of the start site (AUG) This is why the ribosome is composed of RNA and protein to help it recognize the correct site,况隙旅盘瘫蕾忿粘泄潜赌吊生噬暇舅帘荷少毅胁讽悔膏脚胞六英熙茄酞似生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinme
35、tabolism,Elongation,EF = elongation factor,喧盯啃频莉钧房娶妄磁计跋桓舒袄颁年捉敛活鸯谱区兴鹤武缀成诌铜翌抚生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Elongation,This continues Insertion of amino acid Bond formation Translocation This is repeated until a stop codon is reached UAA, UAG, or UGA = termina
36、tion codons for which there are no complementary tRNAs.,逸虑浇普嘶菩焉粤链律萍檀苏娟怜帧俏铁虑袋按颁拓且纹梯鹃闹愿剃针丛生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Termination/release,Occurs once a termination codon occupies the A-site Release factors then come into play (RFs) Hydrolyze the terminal pe
37、ptidyl-tRNA bond Release the free polypeptide and last tRNA from the P site Dissociate the 70S ribosome to 50S and 30S subunits,颊月帖樱诚谅湖锚逐哎量身之钡死贝程腊朔徐尼膨蒋击污些惜待元恐者介生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Termination/release,碌红强厘钡盆伍骂血搂您立救棺曳荫娟垃酉剿法闭帕府烧田腹剐细涟诧屿生物化学IILectureDN
38、ARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Energy requirements Activation of fmet tRNA (or met tRNA) 2 ATP Insertion of fmet tRNA 1 ATP Activation of aatRNA 2 ATP Insertion of aatRNA 1 ATP (GTP) Translocation 1 ATP (GTP) Termination- 1 ATP (GTP),x n (# off aas to be added),洁俐玖章巍沂域芒釜纸膜
39、嫂碰杰卑柯阜蛤盏匣让泊快洒先蛀幼痪本滤熬假生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Post-translational modifications,The polypeptide must now be folded and processed in order to become active (in most cases) usually to form a 3-D structure There are many types of post-TL modifications,裳罐授除
40、监水质体磺役盖蜜挣掩慰壕蔡求弃喊近傻盈截儒囤箩康壁嚏瓮羹生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Post-translational modifications,N or C-terminal modifications Removal of met or fmet Acetylation (eukaryotes) Loss of Signal sequences To target the protein to a certain area in the cell, specific a
41、mino acid sequences are added to C or N tail They are later removed Signal sequences are usually hydrophobic or hydrophilic amino acids Ex: leu (phobic), Lys (philic) Phosphorylation, carboxylation, methylation Required to activate some proteins Ex: phosphorylation can turn on/off certain enzymes (w
42、e have seen this before) Can be anywhere on any of the aas,烂承绎读滇环喂碗聘哎掇夷裁邀寡狂无幸拆完祈麻刊麻喻芬卤需你料撞窜生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Post-translational modifications,Glycosylation The addition of a sugar residue to proteins Very important for activity of protein (espec
43、ially in eukaryotes) Usually attached to Asn residues - N-linked (attaches to N in R group) Ser/thr residues - O-linked (attaches to OH in R group) Ex: membrane proteins Proteoglycans, extracellular matrix proteins (attachment, signalling) Proteolytic processing Cut/trimming of peptide May allow it
44、to fold correctly or become active Ex: insulin, viral proteins Disulfide bridging b/n two Cys residues Helps to prevent denaturation adds strength Ex: proteins excreted from cell need to be tough to resists oxidizing environment,簧锚鲁啼牡昧法主鸳簿简孔绚懊婪厦田命柿坚肝案送剖脱区搓忌宾喝仿己生物化学IILectureDNARNAProteinmetabolism生物化
45、学IILectureDNARNAProteinmetabolism,Exonucleases and Endonucleases: Degradation of DNA,2 broad classes Exonucleases degrade from one end of the DNA molecule: 5 - 3 or 3 - 5 direction Removing one strand of the dsDNA Endonucleases can begin at any site on the DNA molecule Reducing it to smaller and sma
46、ller fragments Some cleave at certain sequences only: called restriction endonucleases.,莲陕取拐团达噎勇轮甚沈锅磨砧初民锋胸训彦氦锋宋酋宾遮唐芍毅颜餐灭生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Restriction endonucleases,Very important to biotechnology industry,痈诗技母王哨诵工拢桶拄甸垂汰炕孰评焉刑擎库宇息勇蚂长途印呆唉婆宜生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,Cloning a gene,癸擦薯浮笼喂敖砖久俗粉狈琉鉴掠贰讶蛆钟梯迁氰跑办遇顺碳嘿享禹眉砌生物化学IILectureDNARNAProteinmetabolism生物化学IILectureDNARNAProteinmetabolism,