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CHAPTER 26 RNA Metabolism. Transcription: DNA-dependent synthesis of RNA RNA processing RNA silencing, RNA-dependent RNA and DNA polymerases. Key topics : . Overview of RNA Function. Ribonucleic acids play three well-understood roles in living cells

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chapter 26 rna metabolism
CHAPTER 26RNA Metabolism
  • Transcription: DNA-dependent synthesis of RNA
  • RNA processing
  • RNA silencing, RNA-dependent RNA and DNA polymerases

Key topics:

overview of rna function
Overview of RNA Function
  • Ribonucleic acids play three well-understood roles in living cells
    • Messenger RNAs encode the amino acid sequences of all the polypeptides found in the cell
    • Transfer RNAs match specific amino acids to triplet codons in mRNA during protein synthesis
    • Ribosomal RNAs are the constituents and catalytic appropriate amino acid
  • Ribonucleic acids play several less-understood functions in eukaryotic cells
    • Micro RNA appears to regulate the expression of genes, possibly via binding to specific nucleotide sequences
  • Ribonucleic acids act as genomic material in viruses
overview of rna metabolism
Overview of RNA Metabolism
  • Ribonucleic acids are synthesized in cells using DNA as a template in a process called the transcription
    • Transcription is tightly regulated in order to control the concentration of each protein in the cell at optimal level
  • Being mainly single stranded, many RNA molecules can fold into compact structures with specific functions
    • Some RNA molecules can act as catalysts (ribozymes), often using metal ions as cofactors
  • Most eukaryotic ribonucleic acids are processed after synthesis
    • Elimination of introns; joining of exons
    • Poly-adenylation of the 3’ end
    • Capping the 5’ end
transcription in e coli
Transcription in E. coli
  • Substrates: Nucleoside triphosphates add to the the 3’ end of the growing RNA strand
  • Template: strand is DNA
  • Enzyme: RNA polymerase
replication vs transcription
Replication vs. Transcription
  • Both add nucleotides via an attack of the 3’ hydroxyl of the growing chain to -phosphorus of nucleoside triphosphates
    • RNA synthesis requires ribonucleoside triphosphates
    • RNA synthesis pairs A with U instead of dA with dT
    • Cis Site = Origins
  • Both require catalysis by a Mg++-dependent enzyme
    • RNA synthesis has lower fidelity
    • RNA synthesis does not require a primer for initiation
    • Cis Sites = Promoters
  • Both require a single strand of DNA as molecular template for building the new strand
both dna strands may encode for proteins
Both DNA Strands may Encode for Proteins
  • Adenovirus is one of the causative agents of common cold
  • Adenovirus has a linear genome
  • Each strand encodes for a number of proteins
rna synthesis is catalyzed by the rna polymerase
RNA Synthesis is Catalyzed by the RNA Polymerase
  • Mg++ on the right coordinates to the -phosphate and stabilizes the negatively charged transition state
movement of rna polymerase causes local supercoiling
Movement of RNA Polymerase Causes Local Supercoiling
  • Positive supercoils (overwound) ahead of the bubble
  • Negative supercoils (underwound) behind the bubble
  • Topoisomerase eliminates positive supercoils
bacterial rna polymerase has at least six subunits
Bacterial RNA Polymerase has at Least Six Subunits
  • Two two  subunits function in assembly and binding to UP elements
  • The  subunit is the main catalytic subunit
  • The ’ subunit is responsible for DNA-binding
  • The  subunit directs enzyme to the promoter
  • The  appears to protect the polymerase from denaturation
promoter consensus sequences in dna
Promoter “Consensus” Sequences in DNA
  • Typical promoters have TATA sequence at -10 base pairs (before the transcription start site - +1)
  • -35 Hexamer TTGACA; Other less common sequence elements in very strong promoters
transcription initiation in e coli
Transcription Initiation in E. coli
  • In the closed complex, the DNA in the promoter region is bound to polymerase but not unwound
  • In the open complex, the two chains in the AT-rich promoter region region are separated
  • subunit leaves before elongation starts
termination rna secondary structure affects processivity
Termination: RNA secondary Structure Affects Processivity
  • RNA polymerase “pausing”
    • Elongation rate reduced.
    • RNA/DNA/Polymerase ternary complex
  • Inverted-repeat sequences
    • Hairpin within the product
    • Or heteroduplex with Nontemplate DNA
  • RNA-DNA hybrid is disrupted
  • Stalling promotes dissociation of the polymerase
eukaryotes contain several distinct polymerases
Eukaryotes Contain Several Distinct Polymerases
  • RNA polymerase I synthesizes pre-ribosomal RNA (precursor for 28S, 18S, and 5.8 rRNAs)
  • RNA polymerase II is responsible for synthesis of mRNA
    • Very fast (500 – 1000 nucleotides / sec)
    • Specifically inhibited by mushroom toxin -amanitin
  • RNA polymerase III makes tRNAs and some small RNA products
  • Plants appear to have RNA polymerase IV that is responsible for the synthesis of small interfering RNAs
  • Mitochondria have their own RNA polymerase
pol ii complex assembly in vitro
Pol II Complex Assembly in vitro
  • Assembly is initiated by interaction of TATA-binding protein (TFIID) with the promoter
  • Helicase activity in TFIIH unwinds DNA at the promoter
  • Kinase activity in TFIIH phosphorylates the polymerase allowing the latter to escape the promoter
rna processing
RNA Processing
  • Almost all newly synthesized RNA molecules (primary transcripts) are processed to some degree in eukaryotic cells
    • The 5’-end is capped w/ methylguanosine
    • Introns are spliced out
    • Poly-A tail is built at the 3’ end
  • Processing is catalyzed by protein-based enzymes and by RNA-based enzymes (ribozymes)
  • Only some prokaryotes have to splice out introns but many process their tRNA precursors
c terminal domain ctd of polymerase ii
C-terminal Domain (CTD) of Polymerase II
  • Repeated 7-mer
    • Tyr-Ser-Pro-Thr-Ser-Pro-Ser
    • Phosphorylated
    • Other RNA processing enzymes loaded via P-CTD as well
four major groups of introns
Four Major Groups of Introns
  • Spliceosomal introns are spliced by splicesomes
    • These are most common introns
    • Frequent in protein-coding regions of eukaryotic genomes
  • Group I and Group II introns are self splicing
    • Interrupt mRNA, tRNA and rRNA genes
    • Found within nuclear, mitochondrial, and chloroplast genomes
    • Common in fungi, algae, and plants, also found in bacteria
    • Group I and Group II differ mainly by the splicing mechanism
  • tRNA introns are spliced by protein-based enzymes
    • Found in certain tRNAs in eukaryotes and archae
    • Primary transcript cleaved by endonuclease
    • Exons are joined by ATP-dependent ligase
introns defined by consensus sequences
Introns Defined by Consensus Sequences
  • Sequences Complementary to Small Nuclear RNAs snRNAs
mrna splicing 1
mRNA Splicing (1)
  • snRNAs live in snRNPs (RibonucleoProtein Particles)
  • Sequential assembly of snRNPs to form the Spliceosome
mrna splicing 2
mRNA Splicing (2)
  • Activation of the internal branchpoint Adenine
  • Attack on the 5’ splice site
  • Lariat Formation
  • Attack of the 5’ Junction on the 3’ Splice site
spliceosome ctd association
Spliceosome/CTD association
  • Spliceosome components also associate with CTD
  • Co transcriptional processing
  • Efficient localization to mRNA
polyadenylation
PolyAdenylation
  • Poly A tail at 3’ end
  • Consensus RNA Cleavage Sequence
  • PAP complex loaded from CTD
    • Endonuclease
    • Poly A Polymerase
      • Not template directed
stable rna synthesis
Stable RNA Synthesis
  • rRNA
    • Synthesized by Pol by
    • Processed I in the nucleolus by snoRNAs
  • tRNA
    • Synthesized as longer precursors
  • Both contain modified bases (post-transcriptional)
micrornas regulate mrna stability and transcription
microRNAs Regulate mRNA Stability and Transcription
  • Complementarity to mRNA
  • Anti-viral and regulatory functions
  • RNA induced Silencing Complex (RISC)
    • Dicer endonuclease
    • RNA helicase
    • Polymerase, Rdp
  • RNA induced Transcriptional Silencing Complex (RITS)
    • Nuclear Complex targets chromatin
transcriptome analysis
TranscriptomeAnalysis
  • mRNA small fraction of total RNA
  • cDNA amplification by primer extension
  • Fluorescent labeling
  • Hybridization to tiled array
  • Expression levels by tissue, developmental stage, disease state,
  • RNA silencing, Chromatin Maintenance
beyond the central dogma
Beyond the Central Dogma

DNA-Directed DNA polymerase

DNA –Directed RNA polymerase

RNA-directed DNA Polymerase

RT, Telomerase

RNA directed RNA Polymerase

SiRNA RDP

retroviral infection
Retroviral Infection
  • RNA Genome
  • Protein Coat
  • Membrane Envelope
  • RT Packed in the virion
  • ReverseTranscription Makes dsDNA
  • Integrase (site specific Recombinase) catalyzes Insertion
retroviral replication
Retroviral Replication
  • Promoters in LTRs
  • RNA transcript encodes a poly protein
  • Poly-Proteins Processed
  • mRNA processed distinct from Genomic RNA
retro elements similar in sequence organization and function
Retro-elements similar in sequence, organization and function
  • Mobile elements replicate infrequently
    • within, not between genomes
telomerase
Telomerase
  • RNA directed DNA polymerase
  • Ribonucleoprotein complex
  • Internal RNA template for DNA Synthesis
  • Inchworm model for synthesis of DNA repeats
chapter 26 summary
Chapter 26: Summary

In this chapter, we learned that:

  • RNA polymerase synthesizes RNA using a strand of DNA as a template and nucleoside triphosphates as substrates
  • The primary RNA transcript in eukaryotes requres processing before it becomes messanger RNA
  • The processing involves capping 5’end with methylguanosine to stabilize the RNA molecule
  • The processing involves splicing out introns
  • Some introns have an amazing ability to carry out their own splicing