第14章regulationofMeetbalism.ppt

1、Chap.14 Metabolism Regulation 代謝網(wǎng)絡(luò)的樞紐物質(zhì)AcCoA 代謝調(diào)節(jié)在3個水平上進(jìn)行細(xì)胞水平，激素水平和神經(jīng)調(diào)節(jié)，最終均以酶水平調(diào)節(jié)為基礎(chǔ)。 14.1 代謝調(diào)節(jié)的相互聯(lián)系 整體網(wǎng)絡(luò)sugar，Lipid，Protein and NA metabolism.密切聯(lián)系，相互作用，相互制約，通過調(diào)控機(jī)制來協(xié)調(diào)。 14.1.1 不同代謝途徑通過共同代謝物形成代謝網(wǎng)絡(luò) 生物分子千萬，不同代謝途徑通過交叉點關(guān)鍵共同中間物溝通。 最關(guān)鍵中間物是 6pG，Pyruvate，AcCoA。次要關(guān)鍵中間物是P-2OH-Aceton，PEP，OAA，-ketoglutarate，p-Ri

2、bose等 。,14.1.2 Sugar和Lipid可以相互轉(zhuǎn)化 糖重要的碳源和能源物質(zhì) 糖代謝中間物 PEP p甘油 AcCoA 脂酰CoA NADPH2 ( From PPP),Fat,膽固醇及其衍生物,脂肪轉(zhuǎn)化成糖因生物種類不同而異。 在動物體內(nèi)，甘油 P-2OH-Aceton 糖（FA不能凈合成糖，關(guān)鍵是丙酮酸生成乙酰CoA為不可逆反應(yīng)）。需要在其它來源的TCA 循環(huán)中間物有機(jī)酸回補(bǔ)時，AcCoA才轉(zhuǎn)變?yōu)镺AA，再經(jīng)糖異生轉(zhuǎn)變?yōu)樘牵?糖異生,在植物和微生物體內(nèi)存在乙醛酸循環(huán) FAT AcCoA 乙醛酸循環(huán) 琥珀酸 OAA 糖（主要發(fā)生在含脂肪種子萌發(fā)時） 14.1.3 糖代謝和蛋白質(zhì)代

3、謝通過TCA循環(huán)相互溝通 糖可轉(zhuǎn)變?yōu)楦鞣NAa 碳骨架，Pyruvate TCA循環(huán) -ketoglutarate，OAA Pyruvate Ala -ketoglutarate Glu OAA Asp TCA循環(huán)的其它中間物和PPP的中間物 -keto acid Aa碳骨架 Aas Protein,糖異生,Energy from Sugar Metabolism,其它Aa可轉(zhuǎn)化為TCA 循環(huán)的中間物，然后合成糖。,14.1.4 TCA循環(huán)是糖、脂肪、蛋白質(zhì)和核酸代謝的樞紐 NA遺傳物質(zhì)，ATP能量通幣，糖基衍生物參與單糖的轉(zhuǎn)變和多糖的合成，CTP參與磷脂合成，GTP參與蛋白質(zhì)合成，cAMP和c

4、GMP第二信使，核苷酸衍生物（如NAD、NADP、CoA、FAD、FMN。在代謝調(diào)節(jié)中起重要作用。 NA合成受其它物質(zhì)的控制。如核酸合成需要酶和多種蛋白質(zhì)因子。Pu和Py合成需要Gln、Gly、Asp、甲酸和NH3為原料。NA分子中的p-Ribose是由PPP提供。NA降解產(chǎn)物的徹底氧化也通過糖代謝途徑。,14.2 Metabolism Regulation p439 14.2.1 生物在4級水平上進(jìn)行代謝調(diào)節(jié) 機(jī)體的結(jié)構(gòu)、代謝和生理功能越復(fù)雜，物質(zhì)代謝的調(diào)節(jié)也更復(fù)雜。高等動物調(diào)節(jié)在4個相互聯(lián)系、彼此協(xié)調(diào)又具有各自特色的層面上進(jìn)行，即神經(jīng)水平、激素水平、細(xì)胞水平和酶水平。 高等動物有高度復(fù)雜各

5、完善的神經(jīng)系統(tǒng)，可根據(jù)內(nèi)外環(huán)境的變化，通過神經(jīng)系統(tǒng)對體內(nèi)各器官的代謝與生理功能進(jìn)行快捷而有效地控制和協(xié)調(diào)，因此神經(jīng)調(diào)節(jié)是最高級水平的調(diào)節(jié)。 激素是內(nèi)分泌細(xì)胞產(chǎn)生的一類微量調(diào)節(jié)物質(zhì)，通過體液運(yùn)輸，作用于一定組織和細(xì)胞，對整體的代謝進(jìn)行綜合調(diào)節(jié)。動物和植物都需要激素調(diào)節(jié)使不同組織細(xì)胞內(nèi)的代謝彼此協(xié)調(diào)。 隨著生物的進(jìn)化和發(fā)展，就整個生物界來說，最基本、最原始的調(diào)節(jié)是細(xì)胞水平的調(diào)節(jié)，是動物、植物和單細(xì)胞,生物所共有。實質(zhì)上，細(xì)胞水平的調(diào)節(jié)是以酶為核心的分子水平的調(diào)節(jié)。 神經(jīng)水平調(diào)節(jié) 激素水平調(diào)節(jié) 細(xì)胞水平調(diào)節(jié) 酶水平調(diào)節(jié),動 物,植 物,微 生 物,代謝調(diào)節(jié)的4個水平,細(xì)胞水平的調(diào)控有3個方面：基因表

6、達(dá)，酶活性（改變關(guān)鍵酶活性）和不同代謝途徑的區(qū)域化。,14.2.2 不同代謝途徑有嚴(yán)格的細(xì)胞定位 a. 細(xì)胞有精細(xì)結(jié)構(gòu)。真核細(xì)胞有內(nèi)膜系統(tǒng)和細(xì)胞器。酶位于不同細(xì)胞器中。 b. 細(xì)胞內(nèi)不同區(qū)域各種代謝物的濃度不同。大多代謝物必須借助膜上的專門運(yùn)載系統(tǒng)從膜的一側(cè)到另一側(cè)。,14.2.3 酶水平調(diào)節(jié)是機(jī)體最基本、最普遍的調(diào)節(jié)方式 調(diào)節(jié)方式：酶含量和酶的活性 p442 酶含量的調(diào)節(jié) 合成速度（主要在基因轉(zhuǎn)錄水平）和降解速度 酶含量不變，通過改變酶的構(gòu)象或結(jié)構(gòu)而改變活性 Activation of proenzyme Allosteric effect Association and deassocia

7、tion p443 Isoenzyme feedback inhibition Covalent Modification two alpha ( ) one beta () one beta prime () and omega () the active enzyme is called the “core enzyme” MW of 450 000 daltons (Da) Sigma factor is 85 000 Da small proteinase factor The core enzyme plus the sigma factor is called the holoen

8、zyme or complete enzyme,The holoenzyme performs 4 functions: Recognizes a promotor on the ds DNA Denatures and unwinds DNA at the promoter Must align itself on the template and transcribe the complete gene Must stop transcription at the terminator Unlike in replication where the total length of the

9、chromosome is replicated in transcription first the gene to be transcribed needs to be identified Transcription can be divided into 3 stages: Initiation, Elongation and Termination,Initiation RNA polymerase must be able to recognize the beginning of a gene so that it knows where to start synthesizin

10、g an mRNA. It is directed to the start site of transcription by one of its subunits affinity to a particular DNA sequence that appears at the beginning of genes. This sequence is called a promoter. It is a unidirectional sequence on one strand of the DNA that tells the RNA polymerase both where to s

11、tart and in which direction (that is, on which strand) to continue synthesis. The bacterial promoter almost always contains some version of the following elements: Promoter recognition : at 10bp region Pribnow box at 35 bp region, these are known asconsensus sequences since they are similar in diff.

12、 Genes in the Upstream same organism,The efficiency and rate of transcription may be directly related to differences in these 2 promoter sequences. Elongation : The RNA polymerase then stretches open the double helix at that point in the DNA and begins synthesis of an RNA strand complementary to one

13、 of the strands of DNA. It is the sigma subunit of RNA polymerase that binds to the promoter and starts the elongation but after adding a few ribonucleotides the subunit dissociates from the holoenzyme and elongation proceeds under the direction of the core enzyme. In E.coli this process proceeds at

14、 the rate of about 50 nucleotides/second at 37c Topoisomerase II handles the template supercoiling,Termination : Following elongation of the entire gene the enzyme encounters a specific sequence that acts as a termination signal. In prokaryotes these sequences are about 40 bp in length and are follo

15、wed by six AT base pairs. These AT base pairs are transcribed in a poly U tail, also two fold symmetry can cause hair pin loops, this type of termination is named as Rho independent termination. Rho dependant terminaton: Rho is a large hexmeric protein that interacts with the growing RNA transcript

16、and disrupts the RNA polymerase/DNA template association In bacteria groups of genes whose products are related are often clustered along the chromosome. They are transcribed contiguously and as a result a long mRNA is produced. Since genes in bacteria are sometimes called cistrons these mRNA are na

17、med a polycistronic. The products of genes transcribed in this fashion are all needed at the same time,Transcription in Eukaryotes The general aspects of the mechanism of the process is the same except : - more than one RNA polymerase - promoter and termination sequences are different - RNA processi

18、ng step The 3 major RNA Polymerases in Eukaryotes RNA poly 1 : found only in the nucleolus; catalayses the synthesis of 28S, 18S, 5.8S rRNA these molecules are important in protein synthesis RNA poly II : found only in the nucleoplasm , catalyses the synthesis of all mRNAs and some small nuclear RNA

19、s (snRNA) RNA poly III: found in nucleoplasm; catalyses tRNA, 5S RNA and other small RNA molecules All eukaryotic RNA polymerases have several sub units (2 large and 4 or more small) In eukaryotes transcription occurs within the nucleus and the RNA transcript is not free to associate with ribosomes

20、prior to the completeion of transcription.,Some differences between prokaryote and eukaryote trascription, in eukaryotes; Initiation and regulation of transcription involve a more extensive interaction between upstream DNA sequences and protein factors involved in stimulating and and initiating . In

21、 addition to promoters, other control units called enhancers may be located in the 5 regulatory region upstream from the initiation point, but they have been found within the gene or even in the 3 downstream region beyond the coding sequence. Maturation of eukaryotic mRNA from the primary RNA transc

22、ript involves many complex stages referred to as “processing”.,Promoters Important DNA sequences lie within this region that initiate transcription by DNA polymerase. Two such elements are present; Goldberg Hogness or TATA box located at 30 (30 bp upstream) from the start point of transcription, the

23、 consensus sequence is a heptanucleotide consisting of A and T residues TATAAAA;( 5-3) similar to pribnow box in prokaryotes. It fixes the site of transcription initiation by facilitating denaturation of the helix. The other element is the CAAT box located at 80, it contains the consensus sequence o

24、f GGCCAATCT; any mutation in the CAAT element, results in a marked reduction in transcription. There may be more than one copy of this GC elements in the promoter. They help bind the RNA polymerase.,Enhancers- important for maximal transcription of a gene; there are no consensus sequences for enhanc

25、ers Two types of enhancers (i) Activator ( activates transcription) (2) Repressors (also called silencer elements) Enhancers may involve : Protein binding to the enhancers DNA loop structures forming between the enhancer and gene sequencers Transcription factors Another kind of specific proteins tha

26、t facilitate initiation of transcription,TF1 for RNA Pol I, TFII for RNA Pol II TFIII for RNA Pol. III Some binds to TATA elements other may bind to CAAT or GC elements. In most cases it binds to RNA Pol and facilitate initiation of transcription An overview of RNA Processing mRNA (messenger RNA) Tr

27、anscribed by structural genes (protein coding genes) contain the coded information for the Amino Acid sequence of a protein. mRNAs have 3 main parts : 5 leader sequence- important for the start of protein synthesis Coding sequence sequence that codes for AA 3 trailor sequence- poly A tail,Typical st

28、ructure of a biologically active mRNA molecule start codon stop codon 5leader sequence coding sequence poly A tail The start codon is AUG and stop codons are UAA, UAG and UGA.The production of biologically active mRNA is different, in prokaryotes and eukaryotes In prok. The mRNA transcript functions

29、 directly in translation. Since theres no nucleus translation begins before mRNA is completely transcribed. In eukaryotes the mRNA transcript must be modified in the nucleus by a series of events called post transcriptional modifications or RNA processing.,5 capping - Involves the addition of a guan

30、ine (7 methyl guanosine) to the terminal 5 nucleotide. A capping enzyme is responsible for the addition and completing the process. The 5 cap is required for the ribosome to bind to the mRNA as the initial step in translation,Addition of 3 poly A tail- This poly A tail is usually about 50-250 bp of

31、adenine in length and there is no DNA template for this tail. (poly A tails are found in most mRNA molecules but not all. Ex. Histones mRNA have no poly A tail. Later the enzyme endonucleases cleaves the molecule at the poly A site to generate 3 OH end. The tail is important for the stability of the

32、 mRNA molecule. In general a eukaryotic mRNA mole. Is longer than the required transcript. The enzyme endonuclease cleaves the molecule at the poly A addition site to generate 3 OH end. Introns and exons Eukaryotic mRNAs often contain long insertions of non-amino acid coding sequences. - These seque

33、nces are transcribed into initial RNA transcript but they are removed before a mature mRNA is developed. - First noticed when researchers found that the nucleotide sequence of some genes (globin genes) were longer than necessary to produce the protein product. Eventually it was found that only a few

34、 genes were with out introns.,Introns (intervening sequences) insertions of non A.A coding sequences of a pre mRNA that are removed in the mature process of mRNA. The genes containing introns are called split genes. The ovalbumin gene of chickens contain about 7 introns. Exons (expressed sequences)

35、is the nucleotide sequence that is translated into an A.A sequence. Both are copied into a primary pre- mRNA transcript. The final mature mRNA is very much short than the initial RNA transcript. Splicing mechanism This is the mechanism by which introns are excised and exons are spliced back together

36、. It appears that somewhat different mechanisms work for diff. Types of RNA and also RNA produced in mitochondria and chloroplasts. During the process, first a molecular complex known as spliceosome forms and Then, after a two-step enzymatic reaction, the intron is removed and two neighboring exons

37、are joined together.The enzyme RNA lygase joins the two exons,The small nuclear RNAs are the most essential component in splicesomes,The RNAs Four different classes of RNA are transcribed mRNA, tRNA, rRNA and small nuclear RNA (SnRNA) The first 3 are found both in prokaryotes and eukaryotes, but SnR

38、NAs are limited only to eukaryotes. Stability- Except for mRNAs others are relatively long lived, mRNA are very short lived Genes that code for mRNAs are structural genes that code for proteins, but for other RNAs it is not structural genes since they produce only a RNA molecule In prokaryotes only

39、one RNA polymerase transcribes all genes, but in eukaryotes 3 different RNA polymerases are involved.,Transcriptional regulation and Metabolism (overview),Jan 15, 2004,I. Transcriptional regulation,As opposed to enzyme regulation By protein binding to DNA to allow or prohibit transcription Negative

40、regulation - protein (repressor) binds between promoter and gene to block transcription Positive regulation - protein (activator) binds upstream of promoter to allow transcription,B. By small environmental signal binding to regulatory protein 1. Induction - small molecule turns ON gene (no matter ho

41、w) 2. Repression - small molecule turns OFF gene (no matter how) EXAMPLES,Lac operon,Negative regulation by induction Lac repressor protein is removed by allolactose inducer,Lac operon,- Negative regulation by induction - Lac repressor protein is removed by allolactose inducer Positive regulation by

42、 induction CAP activator allowed to bind in presence of cAMP,Trp operon,- Negative regulation by repression - Trp repressor protein allowed to bind in presence of co-repressor tryptophan,Trp operon,- Negative regulation by repression - Trp repressor protein allowed to bind in presence of co-represso

43、r tryptophan - “Attenuation” - Leader ensures gene is only ON when trp is too low to make proteins,Trp operon,- Negative regulation by repression - Trp repressor protein allowed to bind in presence of co-repressor tryptophan - “Attenuation” - Leader ensures gene is only ON when trp is too low to mak

44、e proteins - Either loop 1-2, 3-4 or 2-3: 2-3 forms when ribosome stalls due to low trp,Transcription I - DNA to Protein,Transcription II - RNA Polymerase,Transcription is RNA Synthesis using a DNA template Different types of RNA polymerase ATP,CTP,UTP,GTP Condensation Reaction 5 to 3 direction of c

45、hain growth Three part process Initiation Elongation Termination,Transcription III - Prokaryotes,Initiation Promotor Core Polymerase Sigma subunit Elongation Termination Hairpin structure in DNA Rho protein,Transcription IV - Eukaryotes,Initiation Promotor General Transcription Factors Step-wise add

46、itional model Elongation Termination DNA secondary structure,DNA Transcription Defined as the transfer of genetic material from ds template DNA to a ss RNA molecule.,The Central Dogma proposed by James Watson, is that genetic information is transferred; From DNA to DNA through replication during its

47、 transmission from generation to generation. From DNA to RNA to protein during its phenotypic expression in an organism.,Transcription is catalyzed by the enzyme RNA polymerase a single strand of RNA is synthesized using a double stranded DNA molecule as a template. The two strands of the DNA molecu

48、le are separated from one another, exposing the nitrogenous bases. Only one strand is actively used as a template in the transcription process, this is known as the sense strand, or template strand. The complementary DNA strand, the one that is not used, is called the nonsense or antisense strand.,R

49、NA polymerase Is specific for RNA synthesis, Will only use ATP, GTP, CTP, and UTP that contains a ribose sugar Synthsizes in the 5-3 direction DNA template 3 ATACTGGAC-5 RNA product 5 UAUGACCUG-3,Characteristics of RNA polymerase Has no proof reading ability The enzyme has 5 polypeptide subunits; tw

50、o alpha ( ) one beta () one beta prime () and omega () the active enzyme is called the “core enzyme” MW of 450 000 daltons (Da) Sigma factor is 85 000 Da small proteinase factor The core enzyme plus the sigma factor is called the holoenzyme or complete enzyme,The holoenzyme performs 4 functions: Rec

51、ognizes a promotor on the ds DNA Denatures and unwinds DNA at the promoter Must align itself on the template and transcribe the complete gene Must stop transcription at the terminator Unlike in replication where the total length of the chromosome is replicated in transcription first the gene to be t

52、ranscribed needs to be identified Transcription can be divided into 3 stages: Initiation, Elongation and Termination,The efficiency and rate of transcription may be directly related to differences in these 2 promoter sequences. Elongation : The RNA polymerase then stretches open the double helix at

53、that point in the DNA and begins synthesis of an RNA strand complementary to one of the strands of DNA. It is the sigma subunit of RNA polymerase that binds to the promoter and starts the elongation but after adding a few ribonucleotides the subunit dissociates from the holoenzyme and elongation pro

54、ceeds under the direction of the core enzyme. In E.coli this process proceeds at the rate of about 50 nucleotides/second at 37c Topoisomerase II handles the template supercoiling,Termination : Following elongation of the entire gene the enzyme encounters a specific sequence that acts as a terminatio

55、n signal. In prokaryotes these sequences are about 40 bp in length and are followed by six AT base pairs. These AT base pairs are transcribed in a poly U tail, also two fold symmetry can cause hair pin loops, this type of termination is named as Rho independent termination. Rho dependant terminaton:

56、 Rho is a large hexmeric protein that interacts with the growing RNA transcript and disrupts the RNA polymerase/DNA template association In bacteria groups of genes whose products are related are often clustered along the chromosome. They are transcribed contiguously and as a result a long mRNA is p

57、roduced. Since genes in bacteria are sometimes called cistrons these mRNA are named a polycistronic. The products of genes transcribed in this fashion are all needed at the same time,Transcription in Eukaryotes The general aspects of the mechanism of the process is the same except : - more than one

58、RNA polymerase - promoter and termination sequences are different - RNA processing step The 3 major RNA Polymerases in Eukaryotes RNA poly 1 : found only in the nucleolus; catalayses the synthesis of 28S, 18S, 5.8S rRNA these molecules are important in protein synthesis RNA poly II : found only in the nucleoplasm , catalyses the synthesis of all mRNAs and some small nuclear RNAs (snRNA) RNA poly III: found in nucleoplasm; catalyses tRNA, 5S RNA and other small RNA molecules All eukaryotic RNA polymerases have several sub units (2 large and 4 or more small) In eukaryo