Ratz - Transcription
|Gene||The fundamental unit of information in living systems. DNA that encodes a useful RNA. Represent only ~30% of entire genome, about 25,000 genes! Only 1.5% are exons!|
|Direction of reading and writing in Transcription||Polymerase reads 3' to 5' and writes RNA 5' to 3'.|
|Sigma Subunit||Subunit of the sole RNA Polymerase in prokaryotes that recognizes promoter sequences on the DNA. Dissociates from holoenzyme once initiation is complete, elongation begins.|
|Eukaryotic RNA Polymerases|| RNA Pol I - pre-rRNA transcription. Except 5s subunit of ribosome. "Read"|
Pol II - mRNA and snRNA transcription. "My"
Pol III - tRNA and 5S rRNA transcription. "Transcript"
|Prokaryotic Promoters||The "TATA" box around -10 and the -35 sequence. Designated on non-template (coding) strand 5' to 3'.|
|Eukaryotic Promoters||Diverse between and within polymerase types.|
|Transcription Factors||Guide RNA Pol II to the start site in eukaryotes. TBP (TATA Binding Promoter), TFIIB (recruits Pol II), TFIIH (phosphorylates CTD of Pol II.|
|4 Driving Forces for Transcription|| 1. Base stacking|
2. PPi is strong leaving group.
3. Spontaneous hydrolysis of PPi to 2Pi
4. Hydrogen bonding
|Errors in Transcription||1 in 10,000 bases incorrect. No proofreading mechanism.|
|Prokaryotic Ribosomal Subunits||70S is the assembled ribosome. This is broken into 50S and 30S subunits. 50S is composed of 5S rRNA and 23S rRNA. 30S is composed of only 16S rRNA.|
|Eukaryotic Ribosomal Subunits||80S is the assembled ribosome. This is broken into 60S and 40S subunits. 60S is composed of 5S rRNA (the one made by Pol III, the rest by Pol I), 28S rRNA, and 5.8S rRNA. 40S is composed of only 18S rRNA.|
|Endonucleases||Degrade nucleic acids at specific INTERNAL sites.|
|Exonucleases||Clip the ends of nucleic acids.|
|snoRNP's||snoRNA + protein. Involved in processing of pre-rRNA transcript in nucleolus of eukaryotes.|
|Eukaryotic rRNA Processing||One long, primary transcript of 18S, 5.8S, and 28S rRNAs is produced and processed in the nucleolus. Processing involves psuedouridine formation and methylation of 2'-OH groups to protect "keeper" segments. Carried out by snoRNP's.|
|Prokaryotic rRNA Processing||No snoRNP's! Long, primary transcript gets strategic 2'-OH methylations to protect "keeper" segs. Some tRNA is also released along with 16S, 23S, and 5S rRNA's.|
|Where are eukaryotic ribosomes assembled?||Cytosol. 5S rRNA (Pol III) meets others for the first time here. All others are produced (Pol 1), matured, and combined with proteins in the nucleolus. Complex assembly.|
|5 Steps of tRNA Processing to Become "Mature"|| 1. 3' and 5' trimming|
2. 3' CCA base pair extension
3. Intron removal
4. Uracil modifications (dihydro-U, ribo-T, pseudouridine)
5. Cloverleaf 2nd-ary structure
|Arms of tRNA Cloverleaf|| 1. D arm - 1 to 3 dihydro-uracils here|
2. Anticodon arm - 5' spot of anticodon is Wobble position
3. T(Psi)C arm - ribothymidine and pseudouridine (psi) here
4. Amino acid arm - 3' end of tRNA (always adenine) will attach to AA
|Tertiary Structures of tRNA's||No simple, repeating secondary structure. But in 3D, tRNA's do have complex and unique forms, e.g. short, double-helical regions|
|Ribothymidine||Same as T, just attached to a ribose. Not otherwise seen in RNA.|
|Pseudouracil||Uracil that bonds with ribose at the 5 position instead of the 1.|
|Dihydrouridine||Reduced form of Uracil base. No double bond between the carbons in positions 5 and 6.|
|Non Watson-Crick Hydrogen Bonding||In secondary structure of tRNA's. Crucial for tRNA folding.|
|Prokaryotic Post-Transcriptional Processing of mRNA||None! Transcript is translated AS it leaves the transcription apparatus. Multiple proteins can come from single transcript.|
|Eukaryotic Post-Transcriptional Processing of mRNA|| All in the nucleus:|
1. 5' cap
2. 3' poly-A tail
3. Splicing (can occur before or after poly-A tail)
Single mRNA = single protein. But alternative splicing!
|Cap Addition Process||Cap Synthesizing Complex tethered to CTD of Pol II produces cap after ~25 bp transcribed. Cap Binding Complex (CBC) replaces the CSC and holds mRNA onto CTD.|
|What is the cap?||GTP attached to 5' mRNA by 5' to 5' bond with three phosphates. Position 7 of G is methylated. Sometimes 2'-OH's of first 2 nucleotides are methylated.|
|What does the cap do?|| 1. Protects 5' ends against cleavage.|
2. Keeps transcript close to spliceosome scaffold, improving efficiency.
3. Allows binding of translational initiation factors for binding to ribosome.
|Where are polyadenylation and splicing factors located?||CTD|
|What does the poly-A tail do?|| Serves as a binding site for a:|
1. Protein that protects the 3' end of mRNA from exonuclease activity.
2. Protein that interacts with the cap complex to provide initiation of translation.
|4 Classes of Introns|| Group 1 & Group 2 introns are self-splicing|
Group 3 introns = Spliceosome
Group 4 introns within tRNA's
|Ribozyme||RNA with catalytic activity that assists in self-splicing introns.|
|snRNP's||Catalyze splicing in eukaryotic mRNA. The aggregate of catalytic snRNP's makes a Spliceosome.|
|Which snRNA's initiate the spliceosome formation?||U1 and U2 snRNA's. U1 aligns with a 5'-GU at the start of the intron and U2 aligns with an AG-3' site that will become the attacking nucleophile.|
|Assembly of the Spliceosome||U4/U6 and U5 join U1 and U2 (ATP dependent) to form an inactive spliceosome. ATP dependent snRNP rearrangement leads to U6 paired with 5' exon and an active spliceosome.|
|Removal of Intron from Spliceosome||A in the U2 pairing site attacks the G at the start of the U6 site, causing lariat formation. The free 3'-OH of the 5' exon attacks the G at the end of intron, releasing the lariat. No ATP required.|
|Alternative Splicing||Producing different mature mRNA's from pre-RNA's by splicing different exons together. Example is calcitonin gene in humans.|