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Lecture 4

Molecular Cell Biology Lecture 4.docx

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Biological Sciences
Dan Riggs

Molecular Cell Biology Lecture 4 1. RNA polymerase transcribes DNA into RNA, 5’ to 3’, by binding to specific DNA sequences and utilizing phosphate bond energy (derived from hydrolysis of the NTP) to drive elongation. 2. Prokaryotic RNA polymerase consists of several subunits and a single type of 
 polymerase conducts all transcriptional activities. 3. In eukaryotes, three different polymerases are used: Pol I: responsible for synthesis 
 of large (rRNAs); Pol II: mRNAs; Pol III: small RNAs (e.g. tRNA). Each of these 
 RNA polymerases is a multisubunit complex. 4. Consensus sequences exist upstream (5’ to) the gene they control and RNA 
 polymerase interacts and specifically binds to some of these. 5. Most eukaryotic RNAs are synthesized as preRNAs, which are then processed to 
 yield a functional mature RNA. 6. Ribosomal RNA (rRNA) is synthesized at specific sites within the nucleus called the 
 nucleolar organizer region, which contains many tandemly repeated copies of the 
 rRNA genes. 7. Pulse chase experiments were instrumental in deciphering how processing occurs. o Transcription is the process in which a DNA strand provides the information for the synthesis of an RNA strand.  The enzymes responsible for transcription in both prokaryotic and eukaryotic cells are called DNA- dependent RNA polymerases (RNA polymerases)  Enzymes are able to incorporate nucleotides, one at a time, into a strand of RNA whose sequence is complementary to one of the DNA strands, which serves as the template  The steps in the synthesis of RNA include: a. First, the association of the polymerase with the DNA template (interaction between two different macromolecules = proteins + nucleic acids)  The site on the DNA to which an RNA polymerase molecule binds prior to initiating transcription is called the promotor; contains the information that determines which of the two DNA strands is transcribed and the site at which transcription begins  Cellular RNA needs the help of additional proteins called transcription factors which help recognize the promoters b. RNA polymerase moves along the template DNA strand toward its 5’ end. As the polymerase progresses, the DNA is temporarily unwound, and the polymerase assembles a complementary strand of RNA that grows from its 5’ terminus in a 3’ direction.  RNA polymerase catalyzes reaction; RNAn + NTP  RNAn+1 + PPi; in which ribonucleoside triphosphate substrates (NTPs) are cleaved into nucleoside monophosphates as they are polymerized into a covalent chain c. Those reactions leading to the synthesis of nucleic acids and proteins must occur under conditions in which there is virtually no reverse reaction. This condition is met during transcription with the aid of a second reaction  PPi  2 Pi a pyrophosphate. PPi produced in first reaction is hydrolyzed to inorganic phosphate Pi the hydrolysis pyrophosphate releases a large amount of free energy and makes the incorporation of nucleotides essentially irreversible d. As the polymerase moves along the DNA template, it incorporates complementary nucleotides into the growing RNA chain.  Nucleotide is incorporated into the RNA strand if it is able to form a proper base pair with the nucleotide in the DNA strand being transcribed  Consequently, the RNA chain does not remain associated with its template as a DNA-RNA hybrid.  RNA polymerases are apable of incorporating from about 20 to 50 nucleotides into a growing RNA molecule per secnd, and many genes in a cell are transcribed simultaneously by a hundred or more polymerases. e. RNA polymerases are capable of forming prodigiously long RNAs = must remain attached to the DNA over long stretches = processive  Even though polymerases are relatively powerful motors these enzymes do not necessarily move in a steady, continuous fashion but may pause at certain locations along the template or even backtrack before resuming their forward progress o Bacteria, such as E.coli, contain a single type of RNA polymerase composed of five subunits that are tightly associated to form a core enzyme.  If the core enzyme is purified from bacterial cells and added to a solution of bacterial DNA molecules and ribonucleoside triphosphates, the enzyme binds to the DNA and synthesizes RNA  RNA molecules produced are not the same as those found within cells because the core enzyme has attached to random sites in the DNA, sites that it would normally have ignored in vivo.  If a purified accessory polypeptide called sigma factor is added to the RNA polymerase before it attaches to DNA, transcription begins at selected locations  Attachment of sigma factor to the core enzyme increases the enzymes affinity for promoter sites in DNA and decreases its affinity for DNA in general  As a result, the complete enzyme is thought to slide freely along the DNA until it recognizes and binds to a suitable promoter region  X-ray crystallographic analysis of the bacterial RNA polymerase reveals a molecule shaped like a “crab claw” with a pair of mobile pincers (or jaws) enclosing a positively charged internal channel. As the sigma factor interacts with the promoter, the jaws of the enzyme grip the downstream DNA duplex, which resides within the channel  Enzyme then separates (melts) the two DNA strands in the region surrounding the start site  Once 10-12 nucleotides have been successfully incorportated into a growing transcript, the enzyme undergoes a major change in conformation and is transformed into a transcriptional elongation complex that can move progressively along the DNA o Promoters are sites in the DNA that bind RNA polymerase. Bacterial promoters are located in the region of a DNA strand just preceding the initiation site of RNA synthesis  Nucleotide at which transcription is initiated (+1) and the preceding nucleotide (-1)  Portions of DNA preceding the initiation site are said to be upstream  One of these stretches of DNA are similar from one gene to another  One of these stretches is centered at approximately 35 bases upstream from initiation site and typically occurs as the sequence TTGACA (consensus sequence) which indicates that it is the most common version conserved sequence  The second conserved sequence is found approximately 10 bases upstream from the initiation site and occurs at the consensus TATAAT  Portions of DNA succeeding it (toward 5’ end) are said to be downstream o Stigma factor is known as the “housek
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