Jan 28

I. Primary transcripts in prokaryotes
i. Primary transcript: in eukaryotes: the initially copied RNA that’s been transcribed, but hasn’t been post-transcriptionally modified into mRNA
b. Open Reading Frame (ORF) : transcript of bacterial genes that can then serve as mRNA without further processing required
i. ORFs usually contain transcripts for >1 protein often with related functions
ii. Ex: pGlo plasmid has 2 ORFs: it is only the case that GFP is expressed if arabinose operon is expressed; B-lactamase is not on the same frame
II. Eukaryotes: focus on human RNA processing
a. 5’ capping on RNA: capping, methylation of purine(adenine & guanine), and addition of poly-A tail (to 3’ end) protects from viruses
i. Pyrimidines are cytosine, thymine, uracil
b. Introns and exons
i. What are introns? Intervening sequences of stretches of nucleotides within RNA transcription (between coding regions) that must be removed before translation
ii. What are exons? Coding regions of RNA that will be spiced together as introns are taken out
iii. How long are introns? Approximately 9 introns per gene; some are up to hundreds of thousands of bases long
1. Genes are made of 5-10% of exons, >90% introns
iv. Um..why introns? Introns by serving as placeholders, can allow exons to be combined in novel ways  opens the door to evolution of new proteins; especially helps with shuffling gene segments
1. Example: Antibodies: mark foreign molecules for destruction by immune system. Antibody proteins are a result of random antibody-gene exons being patched together
a. Antibody genes: lots of segments separated by introns
c. Coupled processes: transcription and RNA splicing
i. Transcription proteins associate very? closely with proteins and ribozymes involved with removing introns
ii. Increase efficiency of RNA processing
d. Mechanisms of RNA splicing
i. Ribozymes: small RNA molecules with enzymatic activity allowing them to base pair with RNA nucleotides at ends of introns on primary transcript and cut transcript RNA at binding site
III. Intron mutations and exon shuffling
IV. mRNA and cDNA
a. complementary DNA (cDNA)
b. Gene Chip microarrays
c. Quantifying introns
d. rtPCR

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