Lecture 20

• Three representations of DNA double helix

  DNA replication is semiconservative

  two parental strands of DNA must be completely separated in the process of replication

  antiparallel strands are joined by the H bonding of base pairs
• DNA structure

  tetranucleotide DNA sequence contains the four constituent bases of DNA: adenine, cytosine, guanine and thymine

  units all contain deoxyribose and they are all linked by 3’-5’- phosphodiester bonds

  P residue leads to 5' end and oxygen atom leads to 3' end
• Base pairing in RNA and DNA

  Two nucleotides on opposite complementary DNA or RNA strands that are connected via hydrogen bonds
• AT base pair

  formation of two hydrogen bonds

  One H bond occurs between the H atom of the 6-amino group of adenine and a carbonyl O atom of thymine

  second H bond occurs between one of the ring N atoms of adenine and a H atom attached to a ring N atom of thymine

  the distance is about 1.11 nm.
• Three base pairs of DNA

  individual bases are planar, but the base pairs are slightly tilted with respect to each other

  the DNA helix is stabilized by the energy of aromatic stacking between successive bases in the helix and H bonding
• Central dogma of molecular biology

  concerns the flow of info from DNA to RNA to protein

  DNA serves as a template for the synthesis of new DNA and also of RNA, and RNA provides a template for protein synthesis

  A) reverse transcription

  
• Semiconservative replication

  If further generations were generated, there would always be two DNA molecules with one of the original parental strands and one newly synthesized strand. All the rest of the DNA molecules would contain only the newly synthesized strands.
• melting curves for DNA

  As the temperature is raised, the hybridized strands begin to separate

  midpoint of the curve where the DNA strands are 50 percent separated is defined as the melting temperature (Tm)

  At an elevated temperature, the strands will completely separate. Upon cooling, the strands will rehybridize to form a double stranded product
• absorbance of light by DNA is less for double stranded DNA than it is for single stranded DNA
• Stem loop structures

  single stranded DNA or RNA can also exhibit base pairing

  The bases in the stem are complementary and hybridize to form base pairs, the bases in the loop do not
• Base pairing can occur between two distinct DNA strands or between a DNA strand and a complementary RNA strand
• hybridized regions and stem loop structures in ssRNA

  There are long complementary regions that contain occasional mismatches and bubbles. There are also a variety of stem-loop structures that branch off from the main chain
• DNA replication

  process by which parental DNA is copied

  general pattern involves the formation of two copies of progeny DNA from one copy of parental DNA

  process of replication is semi-conservative
• DNA polymerase

  DNA Polymerase is an enzyme complex that is responsible for DNA replication. DNA polymerase catalyzes Phosphodiester bond Formation
• DNA polymerase bond formation

  catalyzes the step-by-step addition of deoxyribonucteotide units to a DNA chain

  new DNA chain is assembled directly on a preexisting DNA template

  reaction catalyzed is: (DNA)n + dNTP -> (DNA)n+1 + PPi

  template can be ss (template DNA must be bound to a primer strand that has a free 3' OH group) or ds (one of the chains must be broken at one or more sites)
• The DNA polymerase reaction also requires all four activated precursors: dATP, dGTP, dTTP, and dCTP, as well as Mg2+
• DNA polymerase reaction

  adds nucleotide units to the 5’ end of the DNA primer strand.

  the deoxynucleotide triphosphate substrates are cleaved to release pyrophosphate and add a deoxynucleotide monophosphate unit to the growing strand of DNA

  added nucleotide is complementary to the template DNA strand
• Mechanism of DNA replication

  5’ end toward the 3’ end

  involves the terminal deoxyribose residue of the primer

  3’ hydroxyl group of the terminal deoxyribose attacks the inmost (alpha) phosphate residue of the incoming deoxynucleotide triphosphate

  phosphodiester bridge is formed, linking the primer to the new nucleotide unit

  3’ hydroxyl group of the new nucleotide unit can then attack the next incoming deoxynucleotide triphosphate
• Three types of RNA are involved in gene expression

  messenger RNA is the template for protein synthesis or translation

  transfer RNA carries amino acids in an activated form to the ribosome for peptide bond formation

  ribosomal RNA constitutes the major component of ribosomes
• Messenger RNA

  An mRNA molecule may be produced for each gene or group of genes that is to be expressed in E. coli.

  A distinct mRNA is produced for each gene in eukaryotes

  The mRNA's comprise a heterogeneous class of molecules.
• In E. coli, the average length of an mRNA molecule is about 1.2 kilobases (kb)
• Transfer RNA

  The sequence in which activated amino acids are polymerized from tRNA carriers is dictated by the mRNA template

  There is at least one kind of tRNA for each of the 20 amino acids

  The tRNA's are relatively small, consisting of about 75 nucleotides (about 25 kd)
• ribosomal RNA

  The rRNA's are relatively large molecules that play both a catalytic + structural role in protein synthesis

  In E. coli, there are three kinds of rRNA, called 23S, 16S, and 5S RNA

  One molecule of each of these species of rRNA is present in each ribosome
• Characteristics of E.coli RNA

  rRNA molecules constitute the largest fraction of cellular RNA (about 80%) and contain about 3700 bases (23S), 1700 bases (16S) and 120 bases (5s).

  tRNAs constitute about 15% of the total RNA and average about 75 bases, making them the smallest RNA molecules

  mRNAs represent about 5% of the total and have an average size of about 1200 bases
• Kilobase

  unit of length equal to 1000 base pairs for ds or ssDNA
• RNA polymerase reaction requires

  A template (ss or ds)

  DNA / RNA hybrids are not an effective template

  Activated precursors - All four ribonucleoside triphosphates (ATP, GTP, UTP, and CTP) are necessary.

  A divalent metal ion to interact with the nucleotides. Either Mg2+ or Mn2+
• Comparison of DNA and RNA synthesis

  synthesis is 5' to 3'

  the mechanism of elongation is similar: the 3' OH group at terminus of the growing chains makes a nucleophilic attack on the innermost phosphate of the incoming nucleoside triphosphate

  synthesis driven forward by hydrolysis of pyrophosphate
• Synthesis of RNA differs from DNA

  RNA polymerase does not require a primer

  RNA polymerase lacks the nuclease capability used by DNA polymerase to excise mismatched nucleotides
• Mechanism of transcription

  virtually identical to DNA replication

  proceeds from 5' to 3'

  involves the terminal ribose residue of the primer

  3' OH group of terminal ribose attacks the inmost P residue of incoming nucleotide triphosphate

  phosphodiester bridge is formed, linking the primer to new nucleotide

  3' OH group of new nucleotide can then attack next incoming deoxynucleotide triphosphate
• The coding strand of DNA is not used for transcription, and has the same sequence (except DNA has T instead of U) as the RNA transcript. The coding strand runs in a parallel direction to the RNA
• The template strand is used for transcription, has the complementary sequence to the RNA transcript, and runs in an antiparallel direction to the RNA
• Consensus sequence

  Transcription is initiated on the DNA template starting downstream from promoter sites that contain sequences

  sequences are similar to each other in base composition and sequence, but an individual promoter sequence may vary from sequence by one or two bases
• transcription promotor sites in prokaryotes and eukaryotes

  Prokaryotes have a TTGACA sequence about 35 bases upstream, and the Pribnow box about 10 bases upstream from the transcription start site

  Eukaryotes have a CAAT box about 75 bases upstream, and a TATA box about 25 bases upstream from the transcription start site
• termination of transcription

  Transcription is terminated by various signals--in E. coli, a stable hairpin followed by a sequence of U's leads to dissociation of the RNA from the template DNA.
• modification of mRNA in eukaryotes

  mRNA’s are synthesized in the nucleus and then transported out into the cytosol where protein synthesis takes place

  mRNA's are modified in the nucleus before being exported

  A nucleotide cap is added to the 5' end and a poly(A) tail is added to the 3' end.
• attachment of an amino acid to a tRNA

  Amino acid specific tRNA's carry and donate activated amino acids during protein synthesis
• aminoacyl-tRNA

  The amino acid is esterified to the 3' OH end of the tRNA

  A triplet anticodon on the tRNA hybridizes to the complementary triplet codon of the mRNA
• triplet codons for protein synthesis

  Amino acids are encoded by groups of three bases (triplets) in mRNA starting from a fixed point

  • 3 nt encode each amino acid
  • code is nonoverlapping and has no punctuation
  • code is degenerate for some amino acids
• The codes UAA, UAG, and UGA are termination signals that stop protein synthesis

  The triplet AUG is a start signal as well as a code for internal methionine residues.
• Genetic code

  The way this matrix works, is that the first letter (5’ position) of the triplet codon is given on the left side of the box. The second letter of the codon is on top of the box, and the third letter (3’ position) is on the right side. For all three letters of the triplet, the bases are arranged in the order UCAG