Genetics 4 - RNA metabolism

• mRNAs : messenger RNA's, code for proteins
• rRNAs- ribosomal RNAs, form the basic structure of the ribosome and catalyse protein synthesis
• tRNAs- transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids
• snRNAs - small nuclear RNAs, function in a variety of nuclear processes, including, the splicing of pre-mRNA
• snoRNAs - small nucleolar RNAs, used to process and chemically modify rRNAs
• scaRNAs - small cajal RNAs, used to modify snoRNAs and snRNAs
• miRNAs - microRNA, regulate gene expression typically by blocking translation of selective mRNAs
• siRNAs - small intefering RNAs, turn off gene expression by directing degradation of selective mRNAs and the establishment of compact chromatin structures.
• other noncoding RNAs - function in diverse cell processes including telomere synthesis, X- chromosome inactivation, and the transport of proteins into the ER.
• Which type of RNA blocks the translation of specific mRNAs?

  miRNA
• sense strand ( aka coding strand)

  • 5' to 3'
  • same as RNA sequence
• antisense strand (aka template strand)

  • 3' -> 5'
  • reverse complement of RNA
  • used as 'Template' for RNA
• Transcription- prokaryotes

  Location - cytoplasm

  process - coupled with translation

  promoters - little variation

  RNA polymerase - one type, 5 subunits (including sigma factor), binds promoters

  Transcription factor- sigma factor

  Termination - Rho factor or RNA hairpin

  RNA structure - Polycistronic - controlled by a single promoter and a single terminator

  Post transcriptional modification - None
• Transcription in prokaryotes stages:

  1. RNA polymerase assembles with sigma factor and Locates a promoter
  2. RNA Polymerase unwinds DNA
  3. transcription begins
  4. 10 nucleotides are added which breaks promoter interaction and triggers elongation
  5. sigma factor is released resulting in more elongation
  6. terminal signal is reached and rho factor (or tau or nusA) or A-U rich RNA hairpins form that destabilises polymerase interaction
  7. RNA is released
• in the genetic structure of gene regions; what does UTR stand for?

  untranslated region
• In prokaryotes which protein locates the RNA polymerase to the correct promoter site?


  sigma factor
• Prokaryotic promoter sites:

  • Transcription start site: start of mRNA transcription
  • -10 pribnow box: essential for transcription intiation
  • -35 region : for high transcription rate
  • allows for sequence variation between strains and between species
• Inhibition of Prokaryotic RNA polymerase

  1. Rifamycins - used against Mycobacterium
  2. Tiacumicins e.g. Fidaxomicin - used against C.diff

  • Bind RNA polymerase
  • used as antibiotics
  • high rate of resistance
• Eukaryotic Transcription

  • Location - Nucleus
  • Process - Translation is separate
  • Promoters - Variation
  • RNA polymerase - Five types (I-V)- 10-20 subunits- Requires transcription factors to bind DNA
  • Transcription Factors - Many
  • Termination - At Sequence (I and III) or random (II)
  • RNA structure - Monocistronic - can encode only one polypeptide per RNA molecule
  • Post transcriptional modification - Yes- 5' Capping, 3' processing Cleavage and polyadenylation and Intron Splicing
• Humans have 3 RNA polymerases (I-III)

  • I - 5.8s, 18s , 28s rRNA
  • II - mRNA requires transcription factors
  • III - tRNA
• Eukaryotic Promoter

  • Transcription Start Site- Start of mRNA transcription
  • TATA Box- In many Eukaryotic promoters- TATA binding protein
  • CAAT box- Site for CAAT-enhancer-binding protein
• Transcription- Eukaryotes

  Intiation

  1. TATA biding Protein binds TATA Box with D and DNA bend induced
  2. B recruited to B recognition element (BRE)3. RNA Polymerase II recruited- With Transcription factors (E, H)

  4. H hydrolysis ATP- Pry apart DNA- Exposes template strand

  5.H phosphorylates RNA polymerase IIn- On CTD- Some Transcription factors released (B, E, H)- Elongation can begin
• Transcription - Eukaryotes

  Elongation

  Ribonucleotides incorporated by RNA polymerase II

  Elongation factors

  • bind RNA polymerase
  • some dislodge histones H2A-H2B dimers

  ATP dependant chromatin remodelling complexes

  • histone modification

  DNA topoisomerase (DNA gyrase in bacteria)

  • removes superhelical tension

  RNA processing

  • 5' capping
  • splicing (remove introns)
  • 3' poly adenylation
• Transcription - Eukaryotes

  Termination

  RNA polymerase I (rRNA)

  • specific site on DNA
  • Transcription termination factor for RNA polymerase I - Blocks RNA polymerase I and releases RNA

  RNARNA Polymerase II(mRNA and more)

  • Continues to transcribe
  • RNA transcript cleaved (Xrn2)
  • In mRNA betweenAAUAAAandGU richsequence
  • Additional Xrn2 cleavage induces transcript termination

  RNA Polymerase III (tRNA and more)

  • 4-7 3′ Uracils
• In eukaryotes, which regions of DNA encode for the final processed RNA transcript?

  Exons, 5'UTR and 3'UTR
• In eukaryotes, which RNA polymerase makes mRNA?

  RNA Polymerase II
• mRNA Modification

  RNA polymerase II

  C-terminal Domain (CTD) "Tail"

  • 52 tandem repeats of 7 amino acids-
  • 2 x Serines per repeat
  • Carries pre-mRNA-processing proteins

  Ser5 phosphorylated

  • Capping proteins bind
  • Cap added when 5′ end passes
  • After capping proteins removed

  Ser2 phosphorylated by elongation kinases

  • initial splicing proteins attach

  Ser5 Dephosphorylated

  • 3′ end processing proteins bind

  Complete dephosphorylated

  • Start next RNA synthesis
• 5′ Capping in eukaryotic modification

  • m7Gp cap
  • 5′-5′ triphosphate linkage with guanine
  • 7-nitrogen of guanine is methylated
• Eukaryotic mRNA Modification - 3′ Polyadenylation

  • After 3′ cleavage
  • ~250 Adenine residues
  • Poly-A Polymerase (PAP)

  Cleavage and polyadenylation specificity factor (CPSF)

  Cleavage stimulation factor (CstF)
• Eukaryotic mRNA Modification - Splicing

  • during transcription
  • removes introns
• Eukaryotic mRNA Modification - Splicing

  • during transcription
  • removes introns
  • alternative splicing

  process of splicing

  • Internal Adenine branch point
  • Branch-point binding protein (BBP)
  • U2 auxiliary factor (U2AF)
  • snRNPs
  • U1 Binds 5′ splice site
  • U2 Binds branch point
  • Triplet U4/U6·U5 – Catalyses the breakage

  Exons ~ 200bases

  Introns ~ 10-10^5 bases
• During Eukaryotic mRNA processing, what is added at the 5’ end of the transcript?

  m7Gp cap
• During Eukaryotic mRNA processing; Which enzyme catalyses the addition of the 3′ tail?

  Poly-A Polymerase
• mRNA Modification - Degradation

  • Deadenylase - Binds 5′ cap, shortens poly-A tail- 3′ to 5′ exonuclease activity

  When Poly-A tail ~30

  • 5’ cap removed → Rapid 5′-to-3′ degradation

  or

  • Rapid 3′-to-5′ degradation

  Note: poly-A tail is responsible for mRNA stability

  • acts as a timer
  • 3′ UTRs are also important – Specific endonucleases. (E.g. Transferrin receptor)
• Eukaryotic RNA Modification - rRNA

  rRNA Processing

  • in the nucleolus
  • Bases modified by small nucleolar ribonucleoproteins (snoRNPs)
  • common modifications: Pseudouridine- uridine isomer & 2′-O-methylated nucleotide
• Eukaryotic RNA Modification - tRNA

  • Transfer RNA Processing
  • In nucleus
  • Synthesised by RNA polymerase III
  • folded first then trimmed from larger precursor
  • if introns are present they are spliced by proteins
  • ~10% of nucleotides modified
  • In cytoplasm, Aminoacyl-tRNA synthetases- Adds amino acid
• microRNA - Synthesis

  • Regulate mRNA stability and translation
  • Humans have >400 different miRNAs
  • Regulate 1/3 of genes

  synthesis

  • RNA polymerase II
  • Capped and polyadenylated
  • Assembled with Argonaute + Others
  • Form a RNA-induced silencing complex (RISC)-Bind target RNAs

  - Induce degradation or reduce translation
• Which eukaryotic initiation factor binds to the5’ cap of mRNA?

  elF4E
• Eukaryotic promoters