1、Chapter 16Transcription and Posttranscriptional ProcessingThe term transcription in molecular biology means RNA biosynthesis.In 1955 Francis Crick hypothesized that there were intermediate molecules which participated in trans- ferring genetic information from DNA to protein.The intermediate molecul
2、e was RNA.In 1958 he put forth the famous central dogma.- -DNA- RNA- protein - Temin 1970In 1961 the DNA dependent RNA polymerase was discovered in E coli and the study on the mechanism of transcription began.In 1982 Thomas Cech discovered that one of the precursor RNA in Tetrahymena chould act as a
3、 catalyst and catalyzed self-splicing. It is a ribozyme. Section 1 DNA dependent RNA synthesisRNA synthesis is catalyzed by DDRP ( or simply RNA polymerase. ) Characteristics of RNA synthesisTemplate DNA ( one strand DNA in dsDNA, template strand )Substrate four kinds of NTPEnzyme DDRP ( no reqireme
4、nt of primer, no proof-reading function )Direction ofRNA chain 5 to 3 synthesisBase pairing GC TA AU rule Inorganic Mg+ Zn+ ion The process of transcription can be divided into three stages: initiation, elongation and termination. see the fig. on the blackboardThe two strands in dsDNA are complement
5、ary.They are the coding strand and the template strand.The template strand works as the template for the synthesis of RNA.The synthesized RNA is complementary to the template strand.Its sequence is the same as that of the coding strand with the exception of the substitution of U for T. eg. 5 CGCATTA
6、ACG 3 coding strand 3 GCGTAATTGC 5 template strand 5 CGCAUUAACG 3 RNA transcript The Reaction( NMP )n + NTP - ( NMP )n+1 +PPi Asymmetric Transcription- -_ - -In dsDNA one strand is coding strand. It is not transcribed.The other strand is template strand. It is transcribed.In a segment of dsDNA the c
7、oding information of genes may be in different strands. RNA polymeraseThere is only one type of RNA pol in prokaryotes while there are three types of RNA pols in eukaryotes. E coli RNA polymeraseE coli RNA pol catalyzes synthesis of all the RNAs ( mRNA , rRNA , tRNA etc ) Structure of E coli RNA pol
8、ymeraseThe holoenzyme of E coli RNA pol contains 2() subunitsThe subunit can be dissociated from the holoenzyme.The RNA pol without subunit is called core enzyme: 2() Function of the Subunit can recognize promoters and initiate transcription. can bind regulatory proteins and control the transcriptio
9、nal . rate. + catalyzes RNA synthesis.There may be subunit. Its function is unknown.There are different kinds of subunits. The most common one is 70. Eukaryotic RNA polymerases RNA pol I RNA pol II RNA pol III_ Product 45SrRNA hnRNA tRNA synthesized ( precursor ( precursor 5SrRNA precursors of 18S,
10、28S of mRNA) snRNA 5.8S rRNAs)_ Sensitivity to no high intermediate -amanite_ Structure of Eukaryotic RNA polsEach type of eukaryotic RNA pols contains two different large subunits and ten odd small subunits.The largest subunit of RNA pol II contains consensus sequence at the carboxyl terminal calle
11、d CTD.CTD is composed of several dozens of heptad ( YSPTSPS ) repeats. CTD FunctionRNA pol II with the unphosphorylated CTD participates in the beginning of transcriptional initiation.During the process of intiation many Ser and some Tyr residues of CTD are phosphorylated.RNA pol II with the phospho
12、rylated CTD fulfills the initia- tion and leaves the promoter.RNA synthesis enters in the stage of elongation. Prokaryotic Transcriptional InitiationProkaryotic RNA pol binds the promoter and initiates transcrip- tion.Promoter : DNA sequence that is usually upstream of a genes coding sequence and th
13、at RNA pol binds and initiates transcrip- tion. subunit can recognize the promoterE coli promoters extend from 70 to +30 of the initiation site (+1)Most of them have two regions of consensus sequence , the-35 region ( TTGACA ) and the -10 region ( Pribnow box TATAAT ). 70 can recognize the consensus
14、 sequence.Some promoters of genes with high transcriptional rate have another consensus region , the AT-rich up-element (-40 to -60 ) which subunit binds.The holoenzyme of RNA pol binds the promoter via subunit. strong promoter / weak promoterIt is the -35 and -10 regions and the distance between th
15、em and the distance between 10 region and the transcriptionnal initiation site , that determines the transcriptional rate.The more similar the 35 and 10 regions of a promoter to the consensus sequences of TTGACA and TATAAT , the stronger affinity it has for RNA pol binding.That results in the higher
16、 transcriptional rate.And vice versa. Process of Prokaryotic Trancriptional InitiationRNA pol recognizes and binds the promoter.That forms a close transcription complex.DNA double helix near 10 region unwinds.That results in an open transcription complex.Transcription begins. A triple-element comple
17、x of DNA,RNA pol and the newly synthesized RNA formsThe formation of the triple-element complex causes con- formation change.RNA pol leaves the promoter and elongation begins.As the elongation begins the subunit dissociates from RNA pol.It is the core enzyme of RNA pol that is responsible for the el
18、ongation. Eukaryotic Transcriptional InitiationEukaryotic RNA pols alone can not initiate transcription.Only with the help of transcription factors can they fulfill the task of initiation.Transcriptional initiation of RNA pol II needs not only the enzyme but also multiple transcription factors.There
19、 are consensus sequences in many of the RNA pol II recognized promoters.They are 30 region ( TATA box ) and +1 region ( the ini- tiation site , initiator , Inr ).The initiation stage of RNA pol II can be divided into two steps : the assembly step and the initiation step. The Assembly StepTBP ( TATA-
20、binding protein ) binds TATA box.TFIIB ( or with TFIIA ) binds TBP and promoter.With the help of TFIIF , RNA pol II- TFIIF complex inter- actes with TFIIB and binds TFIIB and promoter.Then TFIIE and TFIIH join them.RNA pol II and the TFII factors form a close initiation com- plex on the promoter. Th
21、e Initiation StepTFIIH has both the helicase and the kinase activities.It unwinds dsDNA.The close initiation complex becomes the open initiation com- plex.TFIIH also catalyzes phosphorylation of CTD.RNA pol II with phosphorylated CTD initiates transcription. The Initiation Stage of RNA pol I or RNA
22、pol IIIThe initiation stage of RNA pol I or RNA pol III is similar to that of RNA pol II.The transcription factors recognize and bind the promoter.RNA pol I or RNA pol III joins them to form an initiation complex.Initiation begins. Initiation Stage of RNA pol I rDNA ( contains genes of 18 S rRNA, 28
23、 S rRNA and 5.8 S rRNA ) is transcribed by RNA pol I. The product is 45SrRNAThere are two consensus sequences in rDNA promoter : core element ( +1 region ) and UCE ( upstream control element ).Transcription factor UBF binds UCE first.Then transcription factor SL1 binds core element.RNA pol I joins t
24、hem. They form a complex on promoter.Initiation begins.RNA pol III initiates transcription of 5SrRNA gene or tRNA gene.There are internal promoters in such genes.Internal promoter means the promoter within the coding region of the gene. tRNA genes promoter has two consensus sequences : A box and B b
25、ox, while 5SrRNA genes promoter has only one con- sensus sequence : C box. Transcriptional Initiation of 5SrRNA GeneTFIIIA binds C box.TFIIIC and TFIIIB bind TFIIIA.RNA pol III binds them. A complex is formed.RNA pol III initiates transcription. Initiation RNA pol I RNA pol III RNA pol II_ATP requir
26、ement no no yes_ A and B or TATA box core consensus sq. core element C box Inr _ CAAT box upstream element UCE GC box etc_ general TFs SL1 TFIIIA B C various TFIIs_ upstream factors UBF various up- stream factors_ Transcriptional TerminationThere are two types of transcriptional termination in proka
27、ryotes independent and dependent. independent terminationThere are two characteristics of independent termination sq.( terminator ).A segment of GC-rich , self-complementary sq.It is followed by a series of T. eg GCCGCCAGTTCGGCTTGCCGCCTTTTThe RNA synthesized is also self-complementary and forms a st
28、em-loop structure followed by aseries of U. U U 5 GCCGCCAG C CGGCGGUC U U GG U U U 3RNA pol interacts with the structure and stops at the templateThe UA pairs are unstable. The newly synthesized RNA re- leases from the template. Transcription terminates. dependent terminationThe terminator of the de
29、pendent termination is not typical.It contains CA-rich region which factor can recognize. factor is a homo-hexamer protein factor , can bind the newly synthesized RNA and moves to the RNA-DNA hybrid region. factor has helicase activity and unwinds the RNA-DNA helix depending on the energy released f
30、rom ATP hydrolysis. Section 2 Posttranscriptional ProcessingMature eukaryotic mRNA has experienced 5- and 3 end processing and splicing. hnRNA ( or mRNA ) is capped at 5 endMost hnRNA have a cap of 7-methylguanosine triphosphate( m7Gppp-) at the 5 end.It is added to the 5 end of the growing transcri
31、pt of 25-30 nucleotides via 55 triphosphate linkage.The cap protects hnRNA ( and mRNA ) from Rnase attack and participates in the binding of mRNA and ribosome. hnRNA ( or mRNA ) has a poly A tail at the 3endAlmost all of eukaryotic hnRNAs ( or mRNAs ) have a poly A tail of 80-250 nucleotides at the
32、3end.It is enzymatically added to the primary transcript intwo stages. CleavageThe transcript is cleaved 10 to 30 nucleotides past a highly conserved AAUAAA sq ( the polyadenylation signal sq ) and within 50 nucleotides before a GU-rich sq. 10-30nts G -A-AG=Exon1 and exon2 are connected. Lariat form
33、 of intron is re- leased. Splicing is completed.= = G -A-AGThe previously described splicing takes place in the spliceosome.Spliceosome contains snRNPs which are composed of snRNAs and proteins.snRNAs in snRNPs are U-rich, and called URNAs.There are 5 URNAs.The process of splicing in the spliceosome
34、 includes spliceo- some assembly, spliceosome activation and splicingAn eukaryotic gene may direct transcription of different hnRNAs from different promoters or using different poly- adenylation sites.There are two promoters in glucokinase gene.In the liver cell transcription initiates from the seco
35、nd pro- moter which is near the coding sq, while in the pancreatic cell transcription initiates from the first promoter which is upstream located.This is an example that one gene can express different hnRNAs ( and mRNAs ). In the different developmental stages of lymphocyte B 3 end processing of the
36、 hnRNA of chain can use dif- ferent polyadenylation sites and yield different hnRNAS ( and mRNA )This is another example. Alternative Splicing Provides for Different mRNAs from the Same hnRNAThe mechanism of alternative splicing includes the selective inclusion or exclusion of exons, the use of alte
37、rnative 5do- nor or 3 acceptor sites. exon1 exon2 exon3=-=-=hnRNA splicing1 (mRNA1) splicing2 (mRNA2) exon1 exon2 exon2 exon3= = = = splicing3 (mRNA3) exson1 exon2 exon3 = = = hnRNA=-=-=2 donor sites in exon1 2 acceptor sites in exon3 3mRNAs= = = exon 1+2+3= = = part of exon 1+e2+e3= = = e1+e2+ part
38、 of exon 3 rRNA precursor processing requires Rnases prokaryotic 30SrRNA precursor- methylation - Rnases cut - precursors of 23SrRNA, 16SrRNA 5SrRNA and tRNA- Rnases cut - 23SrRNA 16SrRNA , 5SrRNA and tRNA eukaryotic 45SrRNA precursor-methylation -snoRNP mediated processing-precursors of 18SrRNA , 2
39、8SrRNA , 5.8SrRNA -snoRNP mediated processing - 18SrRNA , 28SrRNA , 5.8SrRNA tRNA precursor processing tRNA precursor processing in prokaryotes and eukaryotes are similar.It includes 5end cutting 3end cutting and CCA adding (if there isnt CCA) splicing (if there is intron , splicing takes place via
40、enzymatic cutting and joining) base modification. Self-splicing Catalyzed by RNAIn 1982 T. Cech discovered that the intronof Tetrahymena rRNA precursor could catalyzed self-splicing.The intron that can catalyze self-splicing belongs to the group I intron.Group I intron catalyzes self-splicing with t
41、he help of cofactor guanosine or guanosine phosphateThe self-splicing includes two transesterfication reactions5=-=3 exon 1 exon 25=-=3 G-OH G-OH attacks the first nucleotide at the 5end of the intron.Exon 1 is released.5=OH G-=3Exon 1s 3end attacks the nucleotide at the 3 end of the intron. G-=3 5=
42、OHTwo exons are connected. The intron is released.5= =3 G-OHThere are group II introns , which can also catalyze self- splicing.They catalyze self-splicing just like the previously mentioned lariat-form splicing.It also includes two transesterification reactions but does not take place in spliceosom
43、e and does not require any cofactor.Group I and group II introns are ribozymes. Some mRNAs Undergo EditingCoding information can be changed at the mRNA level byRNA editing.In such cases the coding sq of the mRNA differs from that in the cognat DNA.RNA editing is discovered first in protozoan and med
44、iated by g-RNA.Some nucleotides can be changed in or added into or deleted from the mRNA coding sq by RNA editing.That results in change of the genetic codon and / or the reading frame of the coding sq.The expressed protein is changed.An example of human RNA editing is the apolipoproteinB mRNA.In li
45、ver the single apoB gene is transcribed into an mRNA that directs the synthesis of a 100 Kda protein .In the intestine the same gene directs the synthesis of the primary transcript however acytidine deamination converts a CAA codon in the mRNA to UAA at a single specific site.Rather than encoding gl
46、utamine this codon becomes stop codon and a 48 Kda protein is the result. Section 3 RNA dependent RNA synthesis RNA as genetic materialAll plant viruses, several bacteriophages and many animal viruses have genomes consisting of RNA.There are three types of RNA genomes : dsRNA, ssRNA and two copies o
47、f the same ssRNA. The dsRNA and ssRNA are replicated by RNA replicase( RNA dependent RNA polymerase, RDRP ). In most cases RNA genome is single stranded.There are two subtypes of ssRNA genomes the +ssRNA and the ssRNA.+ssRNA genome functions both as genetic material and mRNA, while ssRNA genome serv
48、es only as genetic material.Viruses with ssRNA genomes use the ssRNA as a template for the synthesis of a complement strand , which can then serve as template in replicating the original strand.Retroviruses have two copies of the same ssRNA as the genome.They carry the reverse transcriptase.It has three enzymatic activities : RDDP , DDDP and Rnase H
