华东理工生物化学chap12.ppt

Transcription:Biosynthesis of RNAs,5th edition by Harvey Lodish,Matthew P.Scott,Paul Matsudaira,and James Darnell,4th edition by Harvey Lodish,Arnold Berk,S.Lawrence Zipursky,and Paul Matsudaira,Lehninger Principles of Biochemistry,Fourth Edition,Biochemistry 5th edition Jeremy M.Berg,Lubert Stryer,and John L.Tymoczko,Outline,Transcription:the reactionTranscription in eukaryotesTranscription initiationTranscription factors:regulation of gene expressionmRNA processingmRNA stabilityGene predictionTranscription in prokaryotes Differences between eukaryotes and prokaryotesRNA polymerase in E.coliOperons Regulation of gene expression in prokaryotes,Fertilized egg,It was in this pub,Eagle pub in Cambridge,James Watson and Francis Crick announced their discovery of DNA on February 28,1953.,1953,1993,“We have discovered the secret of life!”,Transcription,Translation,Central dogma,It was first proposed by Francis Crick in 1958 and re-stated in a Nature paper published in 1970:,Transcription:the reaction,RNA synthesis involves:Separation of the DNA strands;Using one of the DNA strands as template;Synthesis of an RNA molecule in the 5 to 3 direction by RNA polymerase using A,G,C and U;No primers needed;No proofreading;In complementary base pairing,A,T,G,and C on the template DNA strand specify U,A,C,and G,respectively,on the RNA strand being synthesized.,Polyploid chromosomes in Drosophila,If our strands of DNA were stretched out in a line,the 46 chromosomes making up the human genome would extend more than two meters.The human genome contains more than 3.3 billion base pairs but only about 30,000 genes.Question:How can cells find the right sites on the chromosomes to star transcription,how long to go and where to stop?,RNA synthesis,Like all biological polymerization reactions,RNA synthesis takes place in three steps:initiation,elongation and termination.Initiation:to search the genomic DNA for initiation sites,also called promoter sites.(For example,E.coli genomic DNA has about 4.6 million base pairs and 2000 promoters.)Cells must have a mechanism to know where to start the transcription.Although gene expression can be regulated at many steps,the most efficient control of gene expression is at the transcription initiation step.Elongation:to unwind a short stretch of DNA to produce a single-stranded DNA as a template.Using correct ribonucleoside triphosphates as substrates and DNA as template RNA polymerase catalyzes the formation of phosphodiester bonds.This process is repeated many times to produce an RNA molecule of correct length.Termination:to detect termination signals that specify where a transcript ends.,Transcription initiation in eukaryotes,Reaction mixtures contained GTP,CTP,ATP and H3-UTP in Tris-HCl buffer.Four mg of native X.laevis DNA was used as DNA template.Reactions were started by adding protein solutions eluted from a DEAE-column.After 20 min of incubation at 30oC the mixtures were transferred onto a DEAE-cellulose discs followed immersion of the discs in 0.5 M Na2HPO4.The filters were then washed and the radio-activities of the discs were determined.From J.Biol Chem.249:241,1974,Three types of RNA polymerases in eukaryotes,There are three types of RNA polymerases.Each contains two large subunits(220 kDa and 150 kDa each)and 10 to 15 smaller subunits.Each type of RNA polymerase transcribes a different set of RNAs:Type I:for rRNA synthesis(most abundant RNAs)Type II:for mRNA synthesis(protein-coding RNAs)Type III:for tRNA and 5s rRNA synthesis(small RNAs)Therefore,the regulation of gene expression is to control the transcription carried out by type II RNA polymerase at specific locations of the chromosomes.,Three types of RNA polymerases in eukaryotic cells,Factors required for transcription initiation by RNA polymerase II,Proteins required:RNA polymerase II;General transcription factors;Specific transcription factors;Mediators.cis-DNA elements:Promoters;Enhancers.,In vitro transcription:transcription starts at specific sites on the DNA,Top line on the left shows the restriction sites in the region of a virus genome where the transcription-initiation site is located by this nascent-transcript analysis.Different restriction fragments were individually incubated with a nuclear extract and radio-labeled ribonucleosides triphosphates.Transcription begins at the start site and ends when pol II molecule runs off the template.The sizes of the products are analyzed by gel electrophoresis.a-amanitin is a specific pol II inhibitor.,Comparison of nucleotide sequences upstream of transcription start site in vertebrate genes,Comparison of nucleotide sequences upstream of the start site in 60 different vertebrate protein-coding genes.Each sequence was aligned to maximize homology in the region from 35 to 20.The tabulated numbers are the percentage frequency of each base at each position.Maximum homology occurs over a six-base region,referred to as the TATA box,whose consensus sequence is shown at the bottom.The initial base in mRNAs encoded by genes containing a TATA box most frequently is an A.,A functional analysis.The effect of specific point mutations in the mouse beta-globin promoter on the ability of that promoter to initiate transcription.Each line represents the transcription level of a mutant promoter relative to the transcription level of the wild-type promoter.Starting at 106 to+475,a total of 130 single-base mutations were generated.The black dots indicate no mutation was generated.Notice that there are three clusters reduced transcription when mutated.Maniatis et al,Science 1987,236:1237-45.,Certain regions in the promoter is required for successful transcription initiation,Two common elements in the promoter region,Basal elements(near the transcription start,-25 bp)“ATAT box”=TATAAAA(AT-rich DNA is easier to denature than GC-rich DNA)Proximal elements(located upstream,-50 to-200 bp)Cat Box”=CAAT and“GC Box”GGGCGG,Linker scanning mutations to identify transcription-control elements,(Thymidine Kinase),Promoter region of a protein-coding gene:the TATA box and the upstream(proximal)elements,A typical promoter region for a protein-coding eukaryotic gene:The TATA box is about 30 bps upstream of the transcription initiation site.The TATA box binds the general transcription factors which direct RNA Polymerase II to the transcription initiation site.Several upstream promoter elements may bind other transcription activators to facilitate transcription.From Gilbert,Developmental Biology,4th edition,In this experiment,the region between the TATA box and the original start point of transcription was progressively shortened by making small deletions.Each time a deletion was made,the beginning point of transcription was shifted further downstream from the original.This experiment indicates that transcription always begins about 30 bases downstream from the TATA box.,Transcription starts about 30 base pairs downstream of the TATA box,Promoter regions of several genes as examples:not all genes have TATA box,In order to begin transcription,RNA polymerase requires a number of general transcription factors(called TFIIA,TFIIB,and so on).The promoter contains a DNA sequence called the TATA box,which is located 2530 nucleotides away from the site where transcription is initiated.(1)The TATA box is recognized and bound by TATA-binding protein(TBP)which is part of transcription factor TFIID,which then enables the adjacent binding of TFIIB(2).The binding of TFIID caused DNA distortion.(3 some of those from human cells can be replaced in biochemical experiments by the corresponding factors from simple yeasts.,Transcription initiation,Transcription factors:specific gene expression,Many genes are expressed in almost all cells,these genes can be called“house keeping genes”,such as genes related to glucose metabolism.The expression of these genes requires pol II and the so-called general transcription factors,such as TFIIB,E,F,H,etc.Other genes,such as insulin,hemoglobin and interferon-g,are only expressed in specific cell types.The expression of these genes are tightly controlled by specific transcription factors.These transcription factors usually contain two characteristic domains,one DNA-binding domain and one activation domain.The DNA binding domain recognize a specific sequence of DNA on the chromosome and the activation domain control the transcription.More than 2000 transcription factors are encoded in the human genome.Based on the structural features of the DNA binding domains of the specific transcription factors can be divided into different groups.These include helix-turn-helix,zinc finger and leucine zipper,etc.,Functional classification of positive-acting transcription factors.Major functional groups are shown in black;specific examples are illustrated in red.The list of examples is not exhaustive.Brivanlou&Darnell,Science,2002,295:813,Transcription activation:enhancers and transcription activators,Enhancers are DNA sequences that can greatly increase the transcription rate.Transcription activators are proteins that bind the enhancer regions to activate transcription.Major structural motifs in the DNA-binding domains:helix-turn-helix;zinc finger;leucine zipper.,These transcription factors each contains one DNA-binding domain(blue)and one or more activation domains(green).Gal4 and Gcn4 are yeast transcription factors.GR,the glucocorticoid receptor,also contains a hormone-binding domain.SP1 binds to GC-rich promoter elements in a large number of mammalian genes.Lodish et al,Molecular Cell Biology,5th edition.,A transcription factor contains a DNA-binding domain and a activation domain,The 3D structure shows a phage 434 repressor protein complex together with 20 base pairs of DNA(note that phage 434 repressor protein is not a mammalian protein.It is used to show the H-T-H structural features).The repressor protein contains the helix-turn-helix motif.Its recognition helix lies in the major groove of DNA.,A homodimmer of a transcription activator:DNA-binding domain and the leucine zipper From McKinght,Sci Am 1991 April,A zinc finger is a protein domain that can bind to DNA.A zinc finger consists of two antiparallel-strands,and an-helix.One very well explored sub-set of zinc-fingers(the C2H2 class)comprises a pair of cysteine residues in the beta sheets and two histidine residues in the a-helix which are responsible for binding a zinc ion.,Interaction between a C2H2 zinc-finger protein and DNA,Gal4 is a C6 zinc-finger protein.Binding of six cysteine residues to two zinc ions in each monomer forms a compact globular domain that interacts with DNA.It binds DNA as a homodimmer.Two monomers interacting to form a coiled coil structure hold the monomers together.,Interaction between Gal4 protein(a C6 zinc-finger protein)and DNA,High-level transcription can be achieved by binding of a transcription activator to an enhancer,1.Enhancer sequences in the DNA can be a single or multiple copies in either orientation;2.Usually located upstream,but it can be downstream,or even located in the introns;3.Can be several kilobases away from the promoter;4.Silencer elements and repressor factors also exist to suppress gene expression.,An expression construct to specifically express the gene of interest in the developing eye in Drosophila,A,a normal Drosophila eye;B,A cell death-inducing gene is expressed under the control of an eye-specific promoter.The fly is normal except that it does not have the eyes.C,both the cell death gene and a death inhibitor gene are expressed under the same promoter in the eye;D,the cell death inhibitor alone is expressed in the eye.,Expression of a cell death gene under the eye specific promoter,A,C,D,B,Some major signaling pathways that deliver an active transcription factor to the nucleus in response to cell surface receptor binding by an extracellular protein ligand.These factors are activated by polypeptide ligand-receptor interaction at the cell surface and then accumulate in the nucleus to regulate the transcription of target genes.,Transcription elongation and termination,Elongation:the carboxyl terminal domain(CTD)of the largest subunit of RNA Pol II contains 50 repeats of a seven-amino-acid peptide,Tyr-Ser-Pro-Thr-Ser-Pro-Ser(TSPTSPS).As transcription starts and transcribe away from the promoter,the CTD is phosphorylated and the general transcription factors dissociate from the initiation complex.RNA Pol II then synthesizes the primary RNA as it moves along the DNA template.Termination:RNA Pol II terminates at multiple sites in the last exon beyond the poly(A)signal.It can be 0.5 to 2 kb downstream from the poly(A)addition site.The termination may be coupled to the cleavage and polyadenylation process.,The C-Terminal Domain(CTD)of RNA Pol II consists of 52 repeats of the consensus heptapeptide TSPTSPS and serves as a platform for the ordered assembly of the factors responsible for transcription,pre-mRNA 5 capping,splicing,and 3 processing at different stages in the synthesis of the nascent transcript.Orphanides&Reinberg Cell 2002,108:439-451.,The C-Terminal Domain(CTD)of RNA Polymerase II Coordinates Transcription and Pre-mRNA Processing,mRNA processing,The cap:when the RNA polymerase has transcribed only about 2030 nucleotides,the free 5 end of the transcript is capped with 7-methyl guanosine through a 5-5-triphosphate linkage.It is essential for the ribosome to bind to the 5 end of the mRNA.Poly(A)tail:transcription by RNA polymerase II terminates at one of the possible termination sites downstream from the poly(A)site in the final exon.Then the primary RNA is cleaved at the poly(A)addition site which is 20 nucleotides downstream of the polyadenylation signal,AAUAAA.100250 A residues are added to the 3 end.Splicing:the introns are spliced out after the transcription has completed for short mRNAs.For longer ones splicing may occur as transcription is still going.The sequences around the 5 and 3 end of the introns are nearly 100%conserved among vertebrate genes.The introns always begin with GU at 5end and end with AG at the 3end.An A residue located 20-50b from 3end in the intron is essential for splicing reaction to occur.,Processing of mRNA,Overview of RNA processing in eukaryotes using b-globin gene as an example,Structure of the 5 methylated cap of eukaryotic mRNA,The distinguishing chemical features are the 5 5 linkage of 7-methylguanylate to the initial nucleotide of the mRNA molecule and the methyl group on the 2 hydroxyl of the ribose of the first nucleotide(base 1).Both these features occur in all animal cells and in cells of higher plants;yeasts lack the methyl group on base 1.The ribose of the second nucleotide(base 2)also is methylated in vertebrates.,Polyadenylation,Schematic representation of poly(A)signals in animals.AAUAAA:poly(A)signal;Poly(A)site:cleavage site;DSE,U-rich or GU-rich downstream elements;nt,nucleotides.Zhao et al.Microbiol Mol Biol Rev 1999 Jun;63(2):405-45,Polyadenylation at