BSCI222 – Lecture 5 (9/17/13)- RNA Molecules and RNA Processing (Ch. 14 continued)- Beyond the discovery of introns, it was surprising to find that genes can be spliced in different ways. o Normal pattern: remove both intronso Alternative: removes introns and middle Exon, leaving Exon 1 and Exon 3 (multiple proteins from a single gene)o Alternative: multiple 3’ cleavage sites for Polyadenylation. Normally get Exons 1,2, 3, and 4. In the alternative path, get Exons 1, 2, 3, and 5. (No longer have 3’ cleavage site after Exon 4, have one after Exon 6 instead). Again, yields different proteins from same gene.o 5’... many bases… TATA box (autobinding proteins, TF 2D)…. Transcription start site…. Exon 1 (GT to AG)…. Intron… “+2400bp”… Exon 2... etc… Stop codon (TGA)…. Polyadenylation sites (TAATAAA)… 3’ cleavage site.o Other oddities: self-splicing introns and RNA editing RNA sometimes have a catalytic activity (heresy, because thought that only proteins could act as enzymes). Certain RNAs can fold up into a structure that catalyzes the splicing of a big part of that RNA. Loops and stems. Big implications for origin of life.  RNA editing: basic mRNA (with not enough nucleotides to encode that protein) pairs with Guide mRNA (which has some extra bases, comes from a different gene, was transcribed elsewhere) and via editing, the extrabases are inserted into the original transcript. Picture says can be addition, deletion, or alteration of bases. Can completely change the meaning of the transcript, different amino acids. No one really knows how or why it evolved. Another example of how the gene is not collinear with the protein.- Substitution editing: In the liver, unedited mRNA has a CAA codon, making glutamine. But that same mRNA in the intestine, the CAA gets edited to UAA, which is a Stop codon, making a protein only about half as long. Both function in transportation of cholesterol but doing it in different ways, in different tissues. Thus,just because you have a genome sequence, doesn’t mean you have all the information.- END of message RNAs- Transfer RNAs:o The models looks like a T-bone steak. End is twisted/ribboned. Amino acid attachment site (always CCA) on the acceptor arm (3’ arm) and an anticodon of three bases (interacts with a codon in mRNA) (on the round bottom of the T).o Sequence of processing: have a precursor tRNA transcript, has introns that need to be removed, and 3’ and 5’ bits that won’t be used and must be cleaved off. The CCA (common to all tRNAs) is added post-transcription, not in the genome.Finally, individual bases in the tRNA are modified, producing mature tRNA (baseaddition and/or base modification). - Ribosomal RNA:o Ribosomes have multiple RNA components and many proteins associated with them.o Ribosome size (measured in “Svedberg” units, tells you how fast it sediments/settles): bacterial is 70S, eukaryotic is 80S. Each has a large and small subunit. Eukaryotic is larger in all respects. o Ribosomal RNA genes are often found in a repeated complex, including many copies. Repeat array. Humans have about 280 copies of rRNA genes per genome. Sometimes also found in multiple parts of the genome.o E. coli example: 30S: one transcription unit, precursor -> modified, methylation -> mature.o Eukaryotic: same thing, primary transcript -> methylation -> mature.- Small RNAs: (have effect on gene expression)o RNA Interference (RNAi): (bad notes, look up in book) Micro RNA (miRNA): transcribed, produce primary miRNA, gets cleavedto produce a short RNA with a hairpin structure. Stem region gets cut out by an enzyme, and one of the strands of the stem combines with a protein to produce a RNA-induced silencing complex. The RNA provides the sequence specificity for that silencing complex, helps direct that protein toa particular spot in that genome, to a promoter/enhancer/silencer region and block the transcription of a particular gene. miRNA binds to mRNA. Also act as a secondary regulatory network (embryos). Small interfering RNAs (siRNA): chopped up by Dicer, associated with a protein into RNA silencer complex, can either silence the translation or induce the degradation of that RNA.- Review: not every gene has introns, just most. All mRNA has upstream UT, coding region, downstream UT. (UT or something)- Knew that the DNA code had to be a triplet code (4 bases would only give you 16 amino acid possibilities, but there are 20 amino acids). o In the third position of the codon, can get nonstandard pairs (“wobble position”), pairs well enough for the tRNA to bind and decode that message.o First position of the Anticodon (C, G, A, U) -> third position of codon (G, U or C,U, A or G, A U or C).o How many tRNAs do you minimally need? Have 20 amino acids, so at least 20 different tRNAs. But, it turns out that no tRNA can decode all of the codons. Have to have at least 2 because of wobble rules, total of 31 minimum. Typical human has between 170 and 570 genes for tRNA, some of which are repeat arrays. Highly expressed genes often have one set of codons of highly abundant tRNAs (translate very fast), whereas the less expressed use rarer tRNAs.o The genetic code has a structure that minimizes the effects of most mutations. (Table hand out, first go up the left side for first base, over the top for second, down the right for third). Some codon families are completely resistant to mutation in the third position (like Val, all 4 possibilities code for it). Some, like Leucine, will still be okay sometimes if first position gets changed. If you go fromon purine to another, or one pyrimidine to another, those are the most common substitutions and those wind up not changing amino acids in the third position. Many mutations will be silent with respect to expression of amino acids.- Amino acids:o Nonpolar, aliphatic R groups: if interchange bases to stay within this group, won’tchange protein structure. Leucine, Ile, Met, Cal, Ala, and Gly.o Polar, uncharged R groups: Ser, Pro, Thr, Gln, Asn, Cys. Flexibilities even in second base.o Aromatic R groups: entire top row, can change second or third positions and protein structure won’t change. Very likely end up with a protein with at least minimal function.o Positively charged R groupso Negatively charged R groups: Asp, Glu- Universal Genetic Code: find in nucleus of eukaryotes and true for most bacteria. Human mitochondrial code is only different by a handful of codons (own