Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry	Spring 2002
Lecture Notes:: 4 March	© R. Paselk 2002
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DNA Structure and Folding 2

C_ot Curve Analysis

Analysis of c_ot curves

• DNA renaturation curve where
  • plot fraction of single-stranded DNA reannealed vs log c_ot (mol*sec/L)
  • c = concentration of single-stranded DNA at time t
  • c_o = concentration of completely denatured DNA at time 0, t_o
  • Basically DNA reanneals rapidly if short, takes increasing times to anneal as non-repetitive lengths increase in logarithmic fashion (essentially a matter of collision/alignment probability).
  • Follow spectrophotometrically, A₂₆₀ decreases with annealing

When we look at c_ot curves find the c_ot values increase with complexity. thus

• Poly A + Poly U reanneal extremely rapidly.
• Mouse satellite (highly repetitive) DNA anneals very rapidly
• viruses next
• then procaryotes
• then eukaryotes

Analysis of c_ot values indicate that viral and prokaryotic DNA has few or no repeated sequences. On the other hand eukaryotes are quite complex with varying degrees of repetition. Thus a eukaryotic genome will have:

1. Unique sequences coding proteins etc. (approximately 1 copy/haploid genome).
2. Moderately repetitive (< 10⁶ copies/haploid genome).
   1. Occurs in segments of 100 - several thousand repetitions interspersed with larger blocks of unique DNA.
   2. Some specifies repetitive DNA of rRNA, tRNA and histones.
   3. Some is also thought to participate in control.
3. Highly repetitive (>10⁶ copies/haploid genome).
   1. Highly repetitive sequences are clustered at the centromeres, in clusters of nearly identical sequences of up to 10 bp tending to repeat thousands of times. This DNA is isolated as the so-called satellite DNA because it sediments as a distinct satellite band in CsCl gradient as a result of its distinct base composition.
4. Inverted repeats ranging from 100-1000 base pairs.
   1. These renatature with first-order kinetics, indicating self-complementary (inverted) sequences. (Other sequences should renature with second-order kinetics, since they must find each other.) Approximately 2 x 10⁶ copies occur in the human genome.
   2. May be used to align homologous chromosomes during meiosis and to facilitate recombination.

RNA Structure

Three main types of RNA:

• tRNA, or transfer RNA. In the older literature it is also called sRNA for soluble RNA, since its small size makes it more soluble than other types.
  • The classic example is yeast phenylalanine tRNA, tRNA^Phe:
    • tRNA^Phe has 76 bases arranged into a "clover leaf" (in two-dimensional representations, see Fig 26-4 on p 851 of your text) secondary structure characterized by extensive hydrogen bonding interactions, analogous to secondary structures in proteins.
    • 42 of the 76 bases are involved in base pairs to create
      • a "stem" where the amino acid is carried esterified to the 3' hydroxyl group of the terminal adenine (tRNA's always have a CCA sequence at their 3' ends), and three arms, clockwise from the stem,
      • the TyC arm named for the conserved sequence of thymine-pseudouridine-cytosine in the open loop,
      • the anticodon arm named for the anticodon sequence in the loop which recognizes the codon in the mRNA,
      • the D arm named for the conserved dihydrouridine in its loop.
      • There is also a variable arm between the TyC arm and the anticodon arm, so called because it varies considerably (3-21 bases) between deferent varieties of tRNA.
    • Yeast tRNA^Phe has a complex folded, tertiary structure analogous to the tertiary structures of proteins, to give an "L" shaped molecule (Fig 26-6, p 852 of your text).
      • The tertiary structure is held together by non-Watson-Crick base parings between the arms and stacking interactions between bases within and between the stems of the arms (Fig 26-7, p 853 of your text).
      • The bases involved in the tertiary base pairings are mostly invariant, indicating a common tertiary folding pattern for all tRNAs.
      • The overall folded tRNA is compact, with most of the bases, with the exception of the -CCA end and the anticodon, inaccessible to solvent.
  • tRNAs have 15 invariant positions, including the seven noted above, and eight position which are specified as to base type (purine or pyrimidine).
  • tRNAs have many modified bases (up to 25%).
    • As in proteins, tRNAs are made from a standard set of monomers, this time the four bases, A, U, C, G. These bases may then be modified post transcriptionally.
    • Nearly 80 modified bases are known, which have been found at over 60 positions in the tRNA molecule.
• mRNA, or messenger RNA.
  • mRNA is of quite variable length, with no necessary or common secondary or tertiary structures. (Wouldn't expect secondary structures, since the important function is to pass on information to code proteins, and the probability that a protein structure with a 20 letter code giving a given structure will also give a particular structure for a 4 letter code is pretty much a chance affair.
  • mRNA is largely unmodified, with the exception of the addition of polyA strings on the end.
  • Some large and small nuclear RNAs are the result of the initial manufacture of messenger in exon-intron form, and the subsequent removal of small RNA exons for degradation.
• rRNA or ribosomal RNA.
  • rRNA, like tRNA has a definite secondary structure due to hydrogen bonding interactions between bases to make double helical stems.
  • This structure is quite complex due to the large size of rRNA, as shown for the proposed structure of the smaller, 16s, rRNA of E. coli, as seen in Figure 23-23 on p 745 of your text.
  • The overall three-dimensional structure of rRNA is more complex because it is accomplished as a supramolecular ribonucleoprotein structure. In this case the actual 3-D structure results from the interaction of the RNA core and a large group of proteins.
  • Because of the complexity of this structure, and the fundamental importance of ribosomes for life, the rRNA sequence is quite evolutionarily stable, and has found a great deal of use in determining the relationships of organisms at the most fundamental levels: Eubacteria vs. Archbacteria vs. Eukaryotes.

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Last modified 5 March 2002