12Mechanisms of Transcription.docx

1、12Mechanisms of TranscriptionMechanisms of TranscriptionChapter 12Central to our understanding of molecular biology is that the genetic information contained in DNA is copied into RNA.This process of copying is called transcription.A. Four requirements for transcriptionB. Steps in transcriptionC. Tr

2、anscription in EukaryotesA. Four requirements for transcriptionThere are 4 things that are necessary for transcription of DNA: 1. template DNA 2. RNA polymerase 3. promoter 4. 4 rNTPs 1. template DNADNA is double stranded but only one parton one strand is transcribed at a time. This is the template

3、DNA. The template DNA determines the sequence of the ribonucleotide bases in the RNA.What is the name for the enzyme that copies DNA into RNA? RNA polymerase2. RNA PolymeraseThe structure of RNA polymerase has been determined for several species. DNA polymerase was found to be shaped like a hand.RNA

4、 polymerase is shaped like a crab claw.What is a crab?2. RNA PolymeraseThere are 2 forms of bacterial RNA polymerases based on the protein subunits that are present: a. the core enzyme b. the holoenzymeWhat is a holoenzyme?A holoenzyme is a complex of many subunits that has a core enzyme, which has

5、function, but this function is increased by the presence of other protein subunits. a. the core enzyme The RNA polymerase core enzyme is made up of 4 different subunits: 1. two (alpha) subunits 2. one (beta) subunit 3. one (beta prime) subunit 4. one (omega) subunit a. the core enzymeThe core enzyme

6、 can bind DNA and transcribe it into RNA, but it has less activity than the holoenzyme. The core enzyme begins transcription at random points in the DNA (it does not recognize promoters like the holoenzyme.)Unlike DNA polymerase, no primer is required by RNA polymerase.The active site of RNA polymer

7、ase is at the base of the claw. Similar to DNA polymerase, the active site can bind 2 Mg+ ions.b. the holoenzymeThe holoenzyme has the 4 subunits from the core enzyme plus (sigma). (sigma) acts as a specificity factor. It specifically binds to promoter sequences in the DNA. It has 2 (alpha) helices

8、that bind in the major groove. In E. coli the main factor is named 70.What are promoter sequences?A promoter sequence is a signal where transcription should start. At the end of the gene a different DNA sequence signals that transcription should stop. This is called the terminator.The side chains on

9、 sigma 70 bind specifically to certain DNA base pairs at the promoter.The holoenzyme binds to the DNA and then slides along the DNA until it finds a promoter sequence. 3. PromoterThe sigma part of the holoenzyme recognizes a specific sequence called a promoter and this is where transcription begins.

10、3. promoterWhat is different about the DNA sequence at the promoter that is recognized by the subunit of the holoenzyme?Scientists sequenced the DNA at many different promoters and compared the sequences. There were some base pairs that were present at the same place in nearly every promoter.The oth

11、er consensus sequence is at -35 base pairs from the start site and is called the -35 region or -35 element.A consensus sequence is a sequence of nucleotides or amino acids in common between regions of homology in different but related DNA or RNA or protein sequences. Often the sequences of many diff

12、erent genes or species are compared to identify the consensus sequence. Strong promoters have DNA sequences that are close or identical to the consensus sequence for the -10 and -35 boxes. Promoters that differ from the consensus sequences will be weaker-there will be less transcription of the gene.

13、Besides the -10 and -35 regions some strong promoters also have a UP element.This sequence is also recognized by the RNA polymerase. It can increase transcription of a gene by 30 times.Some promoters lack a -35 region and instead have an extended -10 region. Recently scientists have identified a new

14、 element that RNA polymerase binds to.This sequence is called the discriminator and it is located downstream of the -10 region.4. 4 rNTPsIn DNA replication, a deoxynucleotide base pairs to the nucleotide on the template strand. Then a phosphodiester bond is formed by DNA polymerase.Transcription is

15、similar to DNA replication in that the new base, which in this case is a ribonucleotide, base pairs to the nucleotide on the template strand.RNA polymerase forms a phosphodiester bond to attach the new base to the growing RNA. A phosphodiester bond forms between the 3 hydroxyl group and the phosphat

16、e on the new base pair.In transcription the new base pair is a ribonucleic acid: rATP, rUTP, rGTP, rCTPA pyrophosphate is released after the phosphodiester bond is formed.Summary of requirements for transcriptionA. Four requirements for transcription: 1. template DNA 2. RNA polymerase 3. promoter 4.

17、 4 rNTPs Summary of requirements for transcription1. template DNA: DNA that encodes RNA and may be on either strand.2. RNA polymerase a. the core enzyme has 4 different subunits: 1. two (alpha) subunits 2. one (beta) subunit 3. one (beta prime) subunit 4. one (omega) subunit2. RNA polymerase b. the

18、holoenzyme has 5 different subunits: 1. two (alpha) subunits 2. one (beta) subunit 3. one (beta prime) subunit 4. one (omega) subunit 5. one (sigma) subunit (sigma) specifically recognizes promoter DNA.3. promoter Scientists identified the promoter consensus sequence. There is a -10 region and a -35

19、 region. Some promoters sometimes also have a UP element. Others have an extended -10 box or a discriminator.4. 4 rNTPs: rATP, rUTP, rGTP, rCTPB. Steps in TranscriptionThere are three steps in transcription.What are they? 1. Initiation 2. Elongation 3. Termination1. Initiationa. There are 3 steps in

20、 initiation: 1. forming the closed complex 2. forming the open complex 3. addition of the first nucleotides and promoter escape (loss of sigma)1. forming the closed complexRNA polymerase binds to the promoter sequence.This is called the closed complex.1. forming the closed promoter complex RNA polym

21、erase (holoenzyme) binds nonspecifically to DNA until it finds a promoter. Specific binding occurs at promoters and the DNA is bound by .What technique could scientists use to determine what DNA base pairs are bound by RNA polymerase?nuclease protection footprintingNuclease protection footprintingTh

22、e location of protein binding sites on DNA can be determined by nuclease protection footprinting.The protein is bound to the DNA. Then DNA is digested with DNases, but the part that the protein was bound to is protected from digestion.A radioactive molecule (32P) is added to the end of the DNA piece

23、s.Then the DNA is separate on a gel. The gel is placed against a piece of photographic film. Where there is radioactivity, the film will turn black. This is called autoradiography. It is used to visualize the DNA bands. Based on nuclease protection footprinting experiments, it was shown that RNA pol

24、ymerase binds from -44 to +2 on the DNA.Which subunit of RNA polymerase binds to UP elements? How could we determine this?The part of the subunit that binds to the UP element is called CTD (CTD stands for carboxyl terminal domain). A flexible linker connects this to the rest of the subunit. The othe

25、r part of the subunit is called NTD for amino terminal domain. Part of the subunit called the CTD binds to the UP-element.Initially the RNA polymerase binds weakly to the DNA promoter, forming the closed promoter complex. Then it binds more tightly and forms the open complex. 2. forming the open com

26、plexThe DNA strands of the double helix separate. About 14 base pairs around the site of the initiation of transcription open to form a transcription bubble. This is called the open complex.2. forming the open complexAfter the RNA polymerase is bound to a promoter, the hydrogen bonds between some ba

27、ses are broken, opening up the DNA from around base pairs -10 to +3. This is called an open complex. This DNA is usually A-T rich, which makes denaturing the DNA easier. Why?3. addition of the first nucleotides and promoter escapeOnce the DNA is denatured, the RNA polymerase has access to the DNA te

28、mplate and can begin adding base pairs. RNA polymerase does not require a primer like DNA polymerase. At this point the complex is called the initial transcribing complex.The first base pair that is added is usually an adenine. The RNA polymerase binds to the adenine and holds it in place for anothe

29、r ribonucleotide to come and be bound to it.RNA polymerase forms a phosphodiester bond between the adenine and the next nucleotide. Then more nucleotides are added. This complex is not stable and the RNA polymerase often releases the template during the first 10 ribonucleotides. Then it will often b

30、ind and begin again. Short RNA molecules of less than 10 base pairs are produced and released by the RNA polymerase. Then the RNA polymerase begins again without dissociating from the DNA template.promoter escape (loss of sigma)After about 10 nucleotides are added, the RNA polymerase begins to move

31、forward on the template DNA. The (sigma) part of the holoenzyme dissociates. This is called promoter escape. After promoter escape, the elongation part of transcription begins.The (sigma) molecule will bind to another RNA polymerase molecule and begin initiation again. Because it is used many times,

32、 we say it is recycled.The 70 factor makes specific contacts with the DNA at the -10 region and -35 region.The 70 factor is divided into 4 regions. Region 4 forms a helix-turn-helix binding motif that binds to the DNA at the -35 element.The 70 factor binds to the -35 region with a helix-turn-helix motif.The