Lecture 5 Midterm Exam Notes.docx

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Biological Sciences
Dan Riggs

BIOB11 – Lecture 5 – Heterogeneous nuclear RNA (hnRNA) synthesis and processing Vocabulary hnRNA (heterogeneous nuclear RNAs): A large group of RNA molecules that share the following properties: 1. They heave large molecular weights (up to about 80S, or 50,000 nucleotides); 2. they represent many different nucleotide sequences; and 3. Preinitiation Complex: The assembled association of general transcription factors and RNA polymerase, required before transcription of the gene can be initiated. Protein Kinase: An enzyme that transfers phosphate groups to other proteins, often having the effect of regulating the activity of the other proteins. Phosphorylation: the addition of a phosphate group to a protein mRNA cap: found at the 5’ end of protein. 7-methylguanosine molecule that is added in a sort of upside down way of 5' to 5' and with the five prime end remaining a triphosphate so instead of having just one phosphate remaining we have three phosphate groups remaining And an upside down G molecule And this is known as the five Prime cap Poly (A) tail: A string of adenosine residues at the 3’ end of an mRNA added post-transcriptionally. Untranslated Region: Noncoding segments contained at both 5' and 3' ends of mRNAs. Complementary DNA (cDNA): The relationship between the sequences of bases in the two strands of the double helix of DNA. Structural restrictions on the configuration of the bases limits bonding on the two pairs: adenine-thymine and guanine-cytosine. (10.2) Genomic clone: little pieces of DNA which a molecular biologist can put in bacteria to propagate them and the copy or part of the chromosome is referred to as a genomic clone Reverse Transcriptase: An RNA-dependent DNA polymerase. An enzyme that uses RNA as a template to synthesize a complementary strand of DNA. Intron: Those parts of a split gene that correspond to the intervening sequences. Exon: Those parts of a spilt gene that contribute to a mature RNA product. Splice Junction: The 5' and 3' ends of each intron. (11.4) Lecture 5: hnRNA Synthesis and Processing Lecture 5 -rna synthesis RNA synthesis begins as a precursor molecule of 45S in size And gets processed into 28S 18 S and 5.8 S molecules -These are made by RNA polymerase 1 What about mRNAs? -Pulse chase strategy -fig11-17 -They get made in the nucleolus and transferred to the Cytoplasm -Messenger RNAs are synthesized as precursors and are made into a pool of molecules known as heterogeneous nuclear RNA Hetero means different a tremendous number of different molecules of different size because there are so many genes expressed Approximately 30,000 Each making one RNA or more than one RNA. The processing events of a RNA are similar to those of the pulse Chase experiment First label the cells with radioactive phosphorus which gets incorporated to the sugar phosphate backbone of RNA Then Purify RNA and centrifuge Then determine the OD or optimal density which will tell you something about the The total RNA that is in the population And also measure the fractions of radioactivity Which tells you something about the RNA molecules labeled This gives you the following graph -When making the fractions the large molecules come out first and the small molecules come out in the end -Optical density are the blue dots And radioactivity are the red dots -In optical density there are a few hnRNAs but at 28S and 18 S those are the ribosomal RNA -Almost all RNA in eukaryotic cell is due to ribosomal RNA Shortly after the holes in the radio active RNA Then truly HnRNA have a heterogeneous number of sizes -What is going on after 15 minutes is that Chase If you measure what happens to the molecules after three hours Then you find The blue line is almost the same but the red line shows Direct before the more larger molecules but now they have shifted to a smaller sizes which are the messenger RNA That are still Heterogeneous in size But we have lost a lot of the RNA due to initial processing Due to the removal of Intron's -The majority of the RNA synthesized is hnRNA but most of the RNA of the cell is ribosomal RNA Something to keep in mind for the two experiments shown One involving ribosomal RNA and one involving heterogeneous nuclear RNA -Most of the newly synthesized RNA in the cell is HnRNA but note that most of the stable RNA of the Cell is ribosomal RNA -Why? it has to do with half-life Meaning after you make it how long after is it degraded Or destroyed -The half-life of each and hnRNA is usually minutes but the half-life of Ribosomal RNA is days months or years -So even though you are making ribosomal RNA at a slower rate it accumulates more overtime This flowchart is explaining what we will look at throughout the next few weeks very important look at it and learn These steps afforded the cell opportunities to conduct regulation So expression level control can be Affected at different levels The first of which is at the transcription level control The second is at processing and the third is Messenger RNA susceptibility and transport to the cytoplasm and lastly where it has to associate with ribosomes or maybe it doesn't translational level control And after the protein is made it is then subjected to various regulations called post-translational control. In the image the promoter region is upstream from the gene In the previous example of a bacterial enzyme (Of a prokaryotic cell) the sigma factor is an example of a transcription factor and there are consensus sequence at -10 and -35 In eukaryotic cells the transcription factors are those that direct the RNA polymerase to to the proper site for initiation In eukaryotic the conserved sequence is called the Tata box is located about -25 relative to the transcription start site. In figure 11 – 18 The first image shows the start site with the tata box upstream from that The second image shows a protein binding to the Tata box called the TBP which stands for tata binding protein. It bends the DNA and unwinds which creates a structure which allows other molecules to bind Such as transcription factors (TF) And tata box associated factors (taf) TFII means transcription factor for RNA polymerase two and the letter could be ABCD etc. Only when you get all of these factors together only then can RNA polymerase come and bind. So these transcription factors are tethered to RNA Polymerase until A complex called transcription factor TFIIH. TFIIH is a protein kinase and the protein group called kinases transport phosphate groups So it phosphorylates RNA polymerase 2 And when this happens the 52 repeats of 7 amino acids of the carboxyl terminal lose their affinity for RNA polymerase and release A
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