Transcription - RNA Synthesis and Processing Flashcards
RNA Overview
- Messenger RNAs encode the aa sequences of all polypeptides found in the cell, 5% of total RNA, most complex
- Transfer RNAs, match specific amino acids to triplet codons in mRNA during protein synthesis, !15% of total RNA
- Ribosomal RNAs are the constituents and catalytic apporpriate aas, ~80% of total RNA, least complex
Prokaryotic RNA Polymerase
Single RNA Pol synthesizes mRNA, rRNA, and tRNA
- Multisubunit enzyme
- holoenzyme has 5 core subunits of a2BBg plus a sixth called sigma
- Core enzyme is responsible for polymerization, but lacks specificity and can not recognize the promoter
- Sigma allows holoenzyme to recognize promoter regions on the DNA
- Lacks 3’-5’ exonuclease so has high error rate
Operon consists of:
Promoter: site for binding RNA Pol
Operator: binding sites for repressor or activator
Structural gene
Transcription Elongation
- Proceeds in the 5’-3’ direction
- energy from cleavage of phosphate bond and by subsequent hydrolysis of pyrophosphate
- sigma factor dissociates from the complex immediately after elongation egins
- Extremely processive - thousands of nucleotides before it dissociates
- Transcription bubble forms from melting of negative superhelicity in the double-stranded DNA
- RNA transcripit is only base paired with the DNA template in this region (about 15 nt)
Termination of Transcription (p-Independent)
Occurs by protein-independent or dependent mechanisms - both rely on hairpin loop structure
Active signals for termination lie in nascent RNA chain - palindromic GC rich region followed by A-T
- RNA Pol pauses at GC
- dA-rU weak hybrid A-T therefore DNA duplex reforms
- Core RNA Pol has less affinity for dsDNA than ssRNA threfore complex dissociates
An RNA hairpin is formed at a palindromic sequence, reducing the length of the RNA-DNA hybrid
- mRNA is released
Protein-dependent termination
Due to the interaction of a terminator protein Rho (p) with the RNA Pol elognation complex as it pauses at a hairpin loop in nascent transcript.
- p protein is an ATP-dependent RNA-DNA helicase that disrupts the RNA-DNA hybrid, leading to disassembly of the elongation complex
- The p helicase binds to a rut site
- p helicase migrates along the mRNA to the elongating RNA polymerase
- p helicase separates the mRNA from the DNA template
Transcription in Eukaryotes
3 different eukaryotic RNA polymerases each synthesizing a different class of RNA
RNA Polymerase I
Synthesizes pre-ribosomal RNA
RNA Pol II
Synthesis of mRNA
RNA Pol III
makes tRNAs
Post-transcriptional Processing of Precursor mRNA
- 5’ capping (added before synthesis of the primary transcript is complete)
- Cleavage, Polyadenylation, and splicing
5’ Cap
- Enhances stability: protection from nucleases
- Enhances translation efficiency
7-methyl-guanosine added by guanylytransferase
Poly-Adenylation to the 3’ end
- Important for mRNA stabiiity
- Helps in translation
- Added downstream of polyA signal in 3’ UTR. 3’ UTR contains cleavage and polyadenylation signals
- Nascent mRNA is considerably longer than length of RNA preceding poly A site
Splicing
The Removal of Introns
- Having separate portions of gene allow for greater diversity –> different gene products
Mechanism of mRNA splicing in Eukaryotes
Intron is spliced out in the form of a lariat, 2 transesterifications
- 2’OH on Branch Site attacks 5’ splice site on end of first exon
- Newly formed 3’-OH on first exon attacks 3’ splice and exons join = Spliced product
- Lariat form of introon with 2’-5’ bond formed
Splicesomes catalyze reaciton
- Small nuclear RNAs in splicesomes are invovled i nthe precise recognition of splice sites and in the actually splciing mechanism - see slide 23
- Base pairing position snRNPs for assembly of the splicesomes
Alternative mRNA processing
- Alternative cleavage and polyadenylation patterns
- Alternative splicing patterns: two different 3’ splice sites
Different mature mRNAs are produced from the same primary transcript
