SU 4

• Microsomal fraction


  Greatest part of cellular RNA derives from
• Ribosomal RNA (rRNA)


  RNA is a structural component of ribosomes
• Soluble RNA (sRNA)

  RNA in the cytoplasm with a relative low molecular weight
• Transfer RNA (tRNA)


  RNA transports amino acids in the cell for the synthesis of proteins

  
• The question started as to the interaction between the function of DNA as carrier of genetic information and the transfer of genetic information in the cell

  
• This was the difficult question to solve after the structure of DNA was resolved
• Central dogma


  Genetic information flows from one type of nucleic acid to another (e.g. from DNA to RNA), from RNA to protein, but never from protein to a nucleic acid

  
• An RNA molecule that contained genetic information encoded by DNA for protein synthesis could not be isolated, but experiments with the bacteriophage of E. coli provided the first material that directed the thoughts to a specific RNA

  
• The first publication that provided clear experimental data in favour of mRNA was published by Sydney Brenner et al. on May 1961 in nature
• DNA


  One type, one function
• RNA


  Various types with separate functions, main role is information transfer from DNA to protein, also has metabolic functions

  
• RNA


  • Can fold to form complex architectural arrangements for ligand binding
  • Ligand binding activity can lead to catalytic/enzymatic activity

  
• RNA structure


  Single stranded has more conformation possibilities than DNA, can form secondary structures like stem-loop structures, and tertiary structures when single strand loops base pair with distant complementary regions
• Paired regions of RNA can't form B-DNA due to steric hindrance from the 2' OH groups, and instead adopt a conformation similar to A form of DNA

  
• Levels of RNA structure organization

  • Single stranded
  • Contains bases ACG and U (not T)
  • Read 5' to 3'
  • Can form secondary structures like hairpin loops
  • Can form tertiary structures when distant complementary regions base pair

  
• Common structural motifs in RNA

  • Stem-loop structures with loops containing structural motifs like U turns
  • Stems can have bulges/internal loops
  • Stem/loops/bulges + junctions = four basic secondary structural elements

  
• Ribosomal RNA (rRNA)


  Folds into secondary structures due to intramolecular base pairing, forms stem-loop structures with loops containing structural motifs, can form tertiary structures when distant complementary regions base pair, different species have different sedimentation coefficients

  
• Transfer RNA (tRNA)


  Synthesized during transcription, small molecules with a linear sequence of ~85 nucleotides, can fold into secondary structures like a cloverleaf, and tertiary L-shaped structures

  
• Aminoacyl-tRNA synthetase reaction


  1. Aminoacyl-tRNA synthetase matches the amino acid with the tRNA

  2. Anticodon of tRNA base pairs with codon of mRNA to specify the amino acid

  3. Aminoacyl-tRNA synthetase catalyzes the attachment of the amino acid to the 3' end of tRNA

  
• Recognition elements for aminoacyl-tRNA synthetase

  • At least 1 base in the anticodon
  • 1 or more of the three base pairs in the acceptor stem
  • The base at canonical position 73 (unpaired base before CCA)

  
• Central dogma


  3 main RNAs (mRNA, tRNA, rRNA) synthesized from DNA templates via transcription, only mRNA directs protein synthesis via translation

  
• Messenger RNA (mRNA)


  Carries the sequence information for synthesis of a protein, in eukaryotes precursor form is hnRNA which is spliced to yield mature mRNA, rich in poly A tails for efficient translation and stability

  
• mRNA directs the synthesis of proteins, its nucleotide sequence is translated into an amino acid sequence by ribosomes

  
• hnRNA and mRNA


  hnRNA has introns and exons, spliceosome removes introns to produce mature mRNA
• Differences between prokaryotic and eukaryotic mRNA

  • Prokaryotes are polycistronic, ends are naked and unprotected, easily degraded
  • Eukaryotes protect ends with 5' cap and 3' poly A tail, more complex processing

  
• Exon


  Part of an mRNA that consists the information for the synthesis of a part of a protein
• Intron


  Non-coding RNA part of the mRNA that is removed through splicing to form an exon for protein synthesis

  
• Poly-A tail


  Homo-oligonucleotide part that occurs on the 3'-OH-terminal of eukaryotic mRNAs

  
• Synthesis of RNA strand


  Catalyzed by DNA-dependent RNA polymerase, uses ribonucleotide triphosphates as substrates, linked in the order specified by base pairing with DNA template

  
• Cytoplasm


  • Processes are coupled
  • Faster protein production
• Exon


  Part of an mRNA that consists the information for the synthesis of a part of a protein, and the information for a protein as a whole may consist of one or more exons of an mRNA. Together they form a functional unit (an equivalent of the prokaryotic cistron)

  
• Intron


  Non-coding RNA part of the mRNA that is removed from the mRNA through splicing to form an exon for protein synthesis

  
• Synthesis of RNA strand

  1. Catalyzed by DNA-dependant RNA polymerase

  2. Uses ribonucleotide triphosphates as substrates

  3. Linked in the order specified by bp with DNA template

  4. All RNA synthesized via RNA polymerase

  5. Exception is RNA primers, made by primase

  6. 1 RNA polymerase in prokaryotes, several in eukaryotes

  
• Transcription in prokaryotes


  1. Binding of σ subunits and β subunit

  2. Subunit structures (2x α, β, β', σ)

  3. Roles of subunits

  
• RNA-transcription


  1. Binding of RNA polymerase to DNA

  2. Initiation of RNA transcription

  3. Chain elongation

  4. Termination

  
• Binding of RNA polymerase to DNA

  1. Sigma subunit of RNA polymerase recognizes promoter sequence

  2. Polymerase enzyme and promoter sequence: forms closed promoter complex

  3. After closed complex established, RNA polymerase unwinds dsDNA forming open promoter complex

  4. Sigma subunit interacts with non-template strand so bases of template strand open to catalytic site of RNA polymerase

  5. DNA footprinting to identify promoter

  
• Promoters


  • 20-200bp in size
  • Typically consists of a 40bp region on the 5' side of the TSS
  • Contains -10 (Pribnow box) and -35 consensus sequences
  • Sigma subunit binds to these conserved sequences
  • The more closely the -35 sequence to the consensus, the greater the efficiency of transcription

  
• Initiation of polymerization


  1. Duplex to be open so RNA polymerase can access single strand template

  2. Pribnow box rich in A:T sequences for easy melting

  3. RNA polymerase has 2 binding sites for riboNTPs (initiation and elongation)

  4. First nucleotide binds at initiation site, 2nd at elongation site

  
• Chain elongation


  1. Catalysed by core RNA polymerase

  2. RNA polymerase translocates along strand prepares for the next nucleotide

  3. 9-12 residues long, sigma subunit dissociates

  4. dsDNA unwinds constantly so polymerase can access template strand, assisted by topoisomerases

  
• Termination


  1. Intrinsic termination sequences serve as termination sites and signals the end of transcription

  2. Rho termination factor recognizes C rich regions in transcript, moves 5'-3' till it reaches transcription bubble, functions as a helicase unwinding RNA:DNA duplexes