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RNA

Core concept

  • RNA = ribonucleic acid
  • the C\(2'\) position has OH (RNA) instead of H (DNA)

Alkali sensitivity

  • RNA is alkali-sensitive and breaks into fragments
  • DNA is alkali-resistant and does not break into fragments

Major RNA types

rRNA (ribosomal RNA)

  • largest RNA class
  • transcribed in the nucleolus

Biogenesis :

  • 45S rRNA precursor + proteins assembled in nucleolus
  • 5S rRNA is made outside the nucleolus
  • 45S + 5S form a complex

  • trimming + methylation produce:

  • 18S (small subunit)

  • 28S + 5.8S (large subunit) + 5S as a complex
  • large subunit: 60S
  • total ribosome: 80S

mRNA (messenger RNA)

  • transcribed from protein-coding genes
  • the initial transcript is hnRNA (pre-mRNA)

Key features:

  • 5' cap
  • poly(A) tail
  • intron removal (splicing)

Organization :

  • 5' cap, leader, start codon, coding region, stop codon, trailer, poly(A) tail

tRNA (transfer RNA)

  • transfers amino acids to ribosomes

Structure :

  • 5' acceptor stem
  • D loop
  • anticodon loop
  • variable loop
  • T \(\Psi\) C loop
  • acceptor stem 3'

tRNA processing:

  • tRNA precursor has introns that are trimmed
  • nucleotidyl transferase adds CCA (5'→3') to the acceptor stem

Functional elements:

  • D loop:

  • contains modified bases (including dihydrouridine)

  • recognition site for aminoacyl-tRNA synthetase

  • anticodon loop:

  • complementary to mRNA codons

  • variable loop:

  • structural stability

  • T \(\Psi\) C loop:

  • contains thymine, pseudouridine (\(\Psi\)), and cytidine

  • acceptor stem:

  • intra-strand hydrogen bonding

  • forms ester bond to amino acid at the A of the CCA

snRNA (small nuclear RNA)

  • involved in splicing and ligation (intron removal)
  • interacts with splicing proteins and pre-RNA via the spliceosome

microRNA (miRNA)

  • post-transcriptional regulation of gene expression
  • ~22 bp

Mechanism :

  • processed/trimmed miRNA associates with RISC (RNA-induced silencing complex)
  • miRISC searches for complementary mRNA, binds, and promotes mRNA degradation

Transcription: RNA polymerases

RNA polymerase I

  • rRNA

RNA polymerase II

  • mRNA, snRNA, miRNA

RNA polymerase III

  • tRNA, 5S rRNA

Polymerase notes:

  • polymerases lack 5'→3' exonuclease activity
  • highly conserved sequences across species

  • the C-terminal domain (CTD) of RNA Pol II is not conserved

  • contains heptapeptide repeats

  • heavily modified by enzymes that regulate RNA Pol II function

Transcription initiation (RNA Pol II)

Promoter elements

  • consensus sequences / sequence motifs
  • TATA box: TATA(T/A)A
  • CAAT box: GGCCAATCT
  • +1 denotes the first nucleotide in DNA used to build RNA

Distal regulatory elements

  • enhancers:

  • distal, often far upstream

  • affect transcription via DNA folding and transcription factors (AF, TF)

  • silencers:

  • distal elements that repress transcription

General transcription factors (GTFs)

  • recruit RNA Pol II to the promoter

Assembly steps :

  1. TF II D contains the TATA-binding protein (TBP)
  2. TBP binds the consensus sequence and recruits TF II A to stabilize TF II D
  3. TF II B binds TF II D and positions TF II A and TF II D
  4. TF II F brings RNA Pol II to the promoter area
  5. TF II E and TF II H (helicase activity) stabilize RNA Pol II on DNA

Then:

  • pre-initiation complex forms with low basal transcriptional cofactor
  • with topo I
  • ATP hydrolysis drives RNA Pol II conformational change and phosphorylation of CTD
  • transcription factors are released

Directionality:

  • RNA Pol II reads the DNA template strand 3'→5'
  • RNA is synthesized 5'→3'
  • no primer required
  • forms phosphodiester bonds; base pairing via hydrogen bonds

Strand naming:

  • the non-coding strand is also called the coding strand and the sense strand (as noted), because it matches the codons for amino acids

Termination :

  • stops at poly(A) tail region

  • cleavage occurs at poly(A) cleavage site near end of protein-coding genes

  • endonuclease cleavage and “torpedo” RNase model

  • exonuclease degrades the downstream RNA and releases RNA Pol II

Post-transcriptional processing

5' cap

  • 7-methylguanosine
  • linked by a 5'–5' triphosphate bridge

Functions:

  1. protects 5' end from exonucleases
  2. initiates protein synthesis

Poly(A) tail

  • polyadenylation signal is upstream of the poly(A) tail
  • poly(A) polymerase adds adenines to the 3' end

Functions:

  1. protection
  2. mRNA export via poly(A)-binding proteins

Splicing

  • removes introns (non–protein-coding sequences) and ligates exons

Benefits :

  1. alternative splicing
  2. lower mutation burden

Spliceosome components :

  • U1, U2, U4, U5, U6 form the spliceosome
  • introns are excised and exons are joined