RNA synthesis

RNA synthesis

1. RNA types

   • 3 main types of RNAs:
     • mRNA: messenger RNA, codes for proteins.
       • 3-5% total RNA.
     • tRNA: transfer RNA, participates to translation.
     • rRNA: ribosomal RNA, major constituent of ribosomes.
       • There are 4 main rRNAs.
     • Noncoding RNAs serve structural and enzymatic functions

2. Transcription

   • the process of RNA synthesis from a DNA template.
   • Genes can be on either strand of the DNA
     • But rarely overlap
   • Transcription creates supercoiling
     • Topoisomerases release supercoils to allow progression
   • Transcription for a gene starts at its promoter
     • Sequences in the DNA tell RNA polymerases where to start
     • Activators are also required to initiate transcription
       • General transcription factors versus regulatory transcription factors
       • Many of these proteins bind directly to the DNA

3. RNA structure

   • Primary structure of RNA
     • RNA = Ribonucleic Acid
     • 4 major differences between DNA and RNA:
       • Contains ribose (instead of deoxy-ribose)
       • Contains U instead of T
       • RNA is unstable
       • Synthesized as a single strand
   • Secondary structure of RNA
     • Pairing between bases: A = U, G = C
     • Also can rarely have non Watson-Crick pairing, such as G = U.
     • Formation of stem-loop secondary structures by intramolecular base pairing
   • Secondary and tertiary structure of RNA
     • Tertiary structure is the folding of a molecule in three dimensions.

4. Transcription enzymes:RNA polymerases

   • RNA polymerases are enzymes performing RNA synthesis. There are 3 types of RNA polymerases in eukaryotes:
     • RNA polymerase I
       • ribosomal RNA
     • RNA polymerase II
       • protein-encoding genes
     • RNA polymerase III
       • tRNA, small nuclear RNA, and 5S rRNA
   • Transcription requires RNA polymerase
     • Different from DNA synthesis:
       • No primer needed to get started
       • Only one strand of DNA is used as the template
       • Transcript does not remain bound to template
       • Higher error rate
       • Multiple RNA Pol bind on the same gene

5. RNA processing

   • splicing of introns
     • eliminates non coding regions of mRNAs to generate mature mRNA for protein synthesis.
   • capping of 5’ end
     • necessary for stability, binding of mRNA to ribosomes and initiation of translation.
   • polyadenylation of 3’ end
     • part of the termination process (addition of poly-A tail). Determine mRNA stability, helps mRNA nuclear export and translation.
   • Splicing
     • Specific to eukaryotes transcription.
     • Introns are present in the DNA and pre-mRNA, not in mRNA
     • Some mRNA can be spliced in more than one way
       • alternative splicing
     • Splice donor and acceptor sites
       • 2’ OH of branch site A attacks phosphodiester bond on donor site G.
         • Cleavage at the donor site. Formation of lariat.
           • 3’ OH of donor site G attacks phosphodiester bond on acceptor site G freeing the lariat.
             • Lariat is degraded
     • mRNA splicing is performed by the spliceosome
       • Spliceosome: nuclear complex made of about 150 proteins + 5 RNAs.
         • Three functions
           • recognize 5’ donor and branch sites
           • bring sites together
           • catalyzes RNA cleavage
       • SnRNP = SnRNA + proteins
         • snRNAs are small nuclear RNA: U1, U2, U4, U5 and U6.
   • Polyadenylation
     • CstF: Cleavage stimulating factor
     • CTD: Pol II C-terminal domain
     • CPSF: Cleavage and Polyadenylation specific factor
     • PAP: polyadenylate polymerase

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