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BIOB10Y3 (39)
Lecture

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Department
Biological Sciences
Course
BIOB10Y3
Professor
Aarti Ashok
Semester
Summer

Description
How is the gene expressed as traits? [DNA  RNA  Protein]  The relationship b/w genes and proteins was demonstrated by experiments done by Beadle & Tatum in 1940s  Looked at the growth of bread mould, Neurospora Crassa  They hypothesized that if the DNA of this organism were damaged in some way, certain genes may be mutated and hence would not be able to express specific proteins o This would then make it difficult for the mould to grow under conditions where the function of that protein is critical o Example: enzyme working in a specific metabolic pathway -One Gene – One Enzyme Hypothesis  We now know that we can’t just go simply from a gene to make a protein  We require an intermediate known as messenger RNA (mRNA)  The process of making this mRNA from DNA (genes) is called transcription o Carried out by DNA-dependent RNA polymerases o Carried out in the nucleus  mRNAs then exported to cytosol Transcription: from DNA to RNA 1. Go from info storage (DNA in nucleus) to info use (mRNA in cytosol) 2. Amplify synthetic output: several mRNAs can be made from each molecule of DNA; each mRNA can be made into large numbers of proteins/polypeptides Transcription: an overview Transcription (or the synthesis of RNA from DNA) is a cyclic process and can be broken down into 3 stages: 1. Initiation RNA polymerase binds a region of DNA close to the beginning of a gene known as a promoter sequence. -polymerase binds to promoter sequence in duplex DNA [CLOSED complex] -polymerase melts duplex DNA near transcription start site, forming a transcription bubble [OPEN complex] -polymerase catalyzes phosphodiester linkage of two initial rNTPs 2. Elongation Upon promoter binding, a conformational change occurs in the polymerase and upon unwinding of the DNA strands, a complementary RNA strand (which involves linking ribonucleotides by phosphodiester bonds) is synthesized. -polymerase advances 3’5’ down template strand, melting duplex DNA and adding rNTPs to growing RNA -complementary RNA strand moves from 5’3’ 3. Termination RNA polymerase reaches a signal on the DNA that causes an extended pause, the transcript is released from the enzyme, the DNA strands come back together and polymerase floats off to find another promoter. -at transcription stop site, polymerase releases completed RNA and dissociates from DNA  You can see that the mRNA made is complementary to one strand of DNA – called the template strand o It is a copy of info  hence, transcript  Ribonucleoside triphosphates are the building blocks of the mRNA o added on in the order dictated by the template strand o linked by phosphodiester bonds o added on 5’3’ direction  RNA polymerase itself covers about 35bp of DNA o The RNA:DNA hybrid is about 9bp long  Overwinding of the DNA ahead of the polymerase o Positive supercoiling  A more accurate depiction of RNA polymerase action: o DNA makes a sharp turn within the enzyme site o RNA exits out of a separate channel Studying RNA Polymerase  The rate of movement of RNA polymerase can be observed in a fluorescence microscope Transcription in Prokaryotes  One type of RNA polymerase: o 5 subunits = core enzyme  Loose association b/w DNA and core enzyme; RNA chains that are begun are not initiated at proper sites o 5 subunits + Sigma Factor (σ) = haloenzyme  Association of complete enzyme w/ DNA at proper site and opening of double helix  Sigma Factor increases the enzyme’s affinity for promoters o transcription initiates at correct sites  Initiation of transcription is difficult – RNA polymerase may make several attempts Promoters  +1 = site (nucleotide position) at which transcription begins  -35 element = consensus sequence Regulatory sequences required for initiating transcription at the correct site  -10 element = Pribnow box -Upstream = towards 3’ end of template (away from transcription start site) -Downstream = towards 5’ end of template  Once 10-12 nucleotides have been incorporated, the polymerase goes through a conformational change that is known as the ‘transcriptional elongation complex’  Sigma factor is lost following for of this compl
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