Lecture 6 Midterm Exam Notes.docx

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Department
Biological Sciences
Course Code
BIOB11H3
Professor
Dan Riggs

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Lecture 6 consensus sequence: The most common version of a conserved sequence. The TTGACA sequence of a bacteria promoter (known as the -35 element) is an example of a consensus sequence. frameshift: Mutations in which a single base pair is either added to or deleted from the DNA, resulting in an incorrect reading from from the point of mutation through the reminder of the coding sequence. missense mutation: if an insertion or deletion occurs then if it is anything but a multiple of three then it shifts the code and creates a missense protein so it doesn't work snRNP (small nuclear ribonuclear protein particle): RNAs required for the mRNA processing that are small (90 - 300 nucleotides long) and that function in the nucleolus. spliceosome: a macromolecular complex containing a variety of proteins and a number of distinct ribonucleoprotein particles that functions in removal of introns. (11.4) ribozyme (self-splicing RNA): An RNA molecule that functions as a catalyst in a cellular reactions. group 1 intron: found in nuclear RNA of lower eukaryotes, and some organelles. Forms elaborate 3D structure due to intra-strand base-pairing group 2 intron: found in organelles, also self-splicing, but mechanism generates an intermediate called a lariat lariat: loop like structure formed by group 2 introns during transcription branch point A: a very reactive nucleotide which participates in the cleavage event close to the three prime exon magic bullets: can be created to downgrade the overregulation of a specific gene Suppress what is wrong without affecting anything else is called magic bullet siRNA: Small (21-23 nucleotide), double stranded fragments formed when double-stranded RNA initiates the response during RNA silencing. microRNA: Small RNAs (20-23 nucleotides long) that are synthesized from many sites in the genome and involved in inhibiting translation or increasing degradation of complementary mRNAs. (11.5) Lecture 6 RNA splicing and related phenomena What happens if things dont go according to plan Consequences of mis splicing? 1. Accumulation of unspliced dna figure 11-2 If splicing is right then the RNA should be exported to the Cytoplasm where the interact with ribosomes to be Translated But if they are not exported then they are usually degraded Then no protein is produced The second thing that can happen is called frameshift 2.Frame shift This can result in a novel protein And perhaps containing a protein sequence which is coded by an intron During an aberrant splicing part of intron 1 still has not been spliced So if this aberrant protein is transferred to the cytoplasm and translated into a protein and what you get is a protein which's normal function does not work The frames are the triplets set of nucleotides which encode the amino acids If it is anything but a multiple of three then it gets shifted and create a missense protein Missense protein produced so it doesn't work A frameshift Results from the addition or deletion of nucleotide To alter the reading frame of the triplet genetic code If a frameshift occurs due to misplicing of the entire intron then the beginning of the protein will be the same but after that intron all of the nucleotide triplets will differ Causing a missense protein In 1982 Tom Cech discovered that RNA that exhibits Self splicing enzymatic activity-called ribozymes- rna enzyme There are two types of introns that you called group one Intron and group 2 introns In the group one Intron's Part of the protein is complementary to self so to can fold onto its self In group two introns they form something called a lariat Figure 11 – 31 The two blue stripes are exons and the red are Introns There are stems and loops of Introns and the stems are where complementary base pairing happen Something important for self splicing RNA is the Structure called branch point A Which is a very reactive nucleotide which participates in the cleavage event close to the three prime exon. The elaborate structure of the Intron's strategically align the exons Such that the cleavage reactions can take place at both the ends and since they are close together they can be ligated easily. Figure 11 – 31 First because of the way the A is aligned As a result of the three-dimensional structure it becomes very reactive And it catalyzes an attack On the splice donor site(The five prime splice site) And makes a two prime to five prime linkage Which generates a loop structure The second reaction takes the cleeved exon with a three prime hydroxyl group and And catalyzes an attack on the other end of the intron In the end, exon one and Exon two are ligated together and the intron Is cleaved out in the form of a lariat -This process is only found in self splicing RNA such as ribozymes but in higher eukaryotes it is different -that spliceosome mediates RNA processing -Spliceosome is the machine which Cleaves Introns and ligate exons -spliceosomes are also known as snRNP's (snurps) or small nuclear ribonuclear protein particles Figure 11 – 32 Spliceosome mediated RNA splicing for higher eukaryotic organisms -Blue is Exon red is Intron and we have identified branch point A Then comes the U1 snRNP And it binds to the splice donor sequence near the five prime end -Then another snRNP Called u2 binds to the branch point Looking at the small image on the right you can see that U2 has complementary base pairs to the sequence of the intron around the A but a gets left out and the A gets torqued down causing stress On the phosphodiester bonds around A so than it becomes reactive To alleviate the stress. -Other snRNP components come in such as u4 and u6 and in the image it shows that they have interstrand pairing and come in as a complex -When they come in u6 Is going to come in and replace u1 and is strategically placed to act as a ribozyme So it will catalyze the formation of this structure to make the A very reactive structure And to put A in very close proximity to the splice donor sequence A then releases the stress by participating in the cleavage reaction And the exons are ligated back together again See animation links on this!!!!! Figure 11-34 Compare spiliceosome mediated RNA with self-spicing RNA Very important comparing image Figure 11 – 35 -RNA Polymerase two is found at it promoter and is going along transcripting RNA -A 5' cap is added -Intron's are spliced out -A poly a tail is added to the three prime end It would be more convenient to bring this package Of molecules with all the components to Serve as a transcription machine -Package is RNA Polymerase two Which has the Poly a tail which has sites for different molecules to bind Which tag along and do part of the job of RNA processing How do we know this? -One simple technique used to examine RNA Processing is called the RNA Gel blot or northern blot Similar to the process of Insitu hybridization The probe which is something you've radioactively labeled is going to find a sequence In the population of RNA molecules that exist -Population of RNA is millions and so they are separate by gel electrophoresis By size -Then you take a piece of paper and put it on top of that gel electrophoresis and the RNA molecules in the gel will bond to this piece of paper in Exactly the same position that it was in the gel Which is called transferring the RNA to a solid support Or filter So then you have immobilized RNAs on filter -Then you add your radio labeled gene probe to know if it is expressed or not Astrix means radiolabeled -so radiolabeled probe So that radiolabeled probe can only find two and RNA that is complementary t
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