Molecular Genetics Unit IV

• Introns do not contain instructions to code for proteins, while exons do and are transcribed int mRNA transcripts.
• Primary RNA transcripts are mosaics of introns and exons and are spliced.
• Eukaryotic mRNA transcripts have a 5' cap and a poly-A tail
• Splicing cuts out introns and splices together exons.
• The 5' cap is made of modified nucleotides and prevents the 5' end from being recognized by nucleases. This prevents degradation.
• The poly A tail is added to the 3' end of the mRNA to prevent degradation and to allow the mRNA to be transported from the nucleus to the ribosomes for translation.
• Polyadenylation is the addition of a poly(A) tail to the 3' end of a mRNA transcript for mRNA maturation.
• The factors needed for polyadenylation are CstF, CPSF, CF I and II, PAP, and PABP.
• CstF is cleavage stimulation factor and is an RNA-binding protein that helps stabilize the protein complex (CstF and CPSF)
• CPSF is cleavage and polyadenylation specificity factor and is an RNA binding protein.
• CstF and CPSF sit in the "tail" of the polymerase; after the pol synthesizes the poly (A) signal sequence, the protein factors jump onto the newly synthesized mRNA.
• Polyadenylation and termination go hand in hand.
• CF I and II are cleavage factors and endonucleases. They cut the mRNA transcript to free it from the polymerase.
• PAP is poly A polymerase. It adds adenine to mRNA to make the poly (A) tail.
• PABP is polyA binding protein. It helps transport mRNA to cytoplasm from the nucleus and prevents enzymes from degrading mRNA
• After CstF and CPSF jump onto the mRNA at poly-A signal sequence, RNA is cleaved by CF I and II (which are associated with components), leading to an mRNA transcript being released. CstF is not bound to the released mRNA
• Then, PAP attaches to the 3' end and begins adding adenines to form the poly (A) tail.
• PABP binds to the poly (A) tail of mRNA and aid in transport and preventing degradation
• After mRNA has been cleaved from polymerase and has had the poly A tail added, transcription must be terminated
• CoTC site is the cotranscriptional cleavage site
• When the CoTC site is transcribed, the mRNA folds up and causes self-cleavage
• 2 important sequence elements for transcription are the poly A signal sequence and the CoTC site
• The CoTC site is a a ribozyme, an RNA molecule with catalytic activity
• When mRNA transcript is cut, Xrn2 can jump onto transcript
• Xrn2 is a 5'->3' exonuclease that "eats" its way up mRNA to polymerase, and knocks Pol off the DNA, causing termination of transcription
• The 3 enzymes involved in addition of the 5' cap are RNA triphosphatase, guanylyl transferase, and methyl transferase
• 1. RNA triphosphotase removes a phosphate group from the end nucleotide of the 5' end
• 2. Guanylyl transferase adds a G-containing nucleotide
• Methyl transferase transfers a methyl group to 2 locations in the 5' cap: the guanine of the 5 cap, and the first nucleotide of the mRNA
• the 5' cap is added very early on in transcription to protect the mRNA as its being elongated
• The 5' end is unrecognizable because the nucleotides are added in the opposite orientation to all other mRNA nucleotides
• The 5' cap is added before the 3' poly (A) tail
• The luciferase experiments showed how important a cap and a tail is for mRNA protection and translatability. Its much more efficient!
• The cap and tail also plays a role in transport and helping in the splicing process
• The luciferase experiments showed that the half life of mRNA is signficantly increased when it has BOTH a cap and a tail. Only having one did not make a huge difference
• There was a significant increase in luciferase production in mRNA with 5'cap, but especially BOTH a cap and tail
• 5' and 3' post transcriptional mods are "nontemplate additions"
• Exons are short for expressed codons while introns are interrupting codos
• The primary transcript is the mRNA molecule with both exons and introns
• premRNA, immature mRNAs, and hn mRNA (heteronuclein) are other terms for primary transcript