Nucleic acids

Chemistry 256

Nucleic acids = polynucleotide

• The term includes both DNA (deoxyribonucleic acid) and RNA (ribonucleic acid)

• Polymers of nucleotides• Nucleotide = nitrogenous base linked to a

sugar linked to at least one phosphate

The nitrogenous base distinguishes the molecules; there are only five “interesting” ones

Note the numbering system of atoms in the structures; these are not-prime (as opposed to the primed numbers in the ribose moiety).

Sugar = ribose

• “Deoxy-” refers to the 2’ carbon does not have a hydroxy group. The carbons on the ribose are “primed” because the nitrogenous base’s carbons are considered the primary chain on the molecule.

Complete nucleotide• The 3’ and 5’ carbons’ hydroxy groups are the

attachment points for phosphate groups.

The phosphates link the nucleosides together

• Phosphodiester bonds.• Like proteins, the polymer

is asymmetric.• The convention is to read

the sequence of nucleosides from the 5’ end (the end with the free phosphate on the 5’ carbon) to the 3’ end.

• Reflects how enzymes “read” the sequence.

Schematic nucleic acid polymer representation• Nucleosides = ribose + base; A = adenosine, C = cytidine, G =

guanosine, T = thymidine• Nucleotides = ribose + base + phosphates; A = adenine, C =

cytosine, G = guanine, T = thymine (these are actually the base names, but hardly anyone uses the full nucleotide name, like cytidine monophosphate (CMP))

In addition, U = uracil (for RNA – no T in RNA)

Brief history of DNA discovery

• Erwin Chargaff (Columbia University) – “The Separation and Quantitative Estimation of Purines and Pyrimidines in Minute Amounts”, J. Biol. Chem.(1948) found that the number of A nucleotides in a sequence of DNA equals the number of T nucleotides; similarly, # of C = # of G (“Chargaff’s rules”).

• However, percentage of A+T does not equal percentage of C+G; for mammals, % G+C is about 39 – 46%.

Double helix

• Though James Watson and Francis Crick (Cambridge University) publish “A Structure for Deoxyribose Nucleic Acid” in Nature (1953) and are credited with coming up with the double-helical structure of DNA, they benefitted from many researchers’ work.

Tautomeric form of bases

• Jerry Donahue (Cambridge) found that the bases can exist as keto or enol tautomers, but the bases in DNA only exist as keto tautomers.

• Led to the idea of hydrogen bonding between two DNA strands.

X-ray crystallography• Maurice Wilkins (who

won the 1962 Nobel Prize, along with Watson and Crick), Alex Stokes and Herbert Wilson (King’s College), and Rosalind Franklin and Raymond Gosling (King’s College) publish simultaneous papers in Nature (1953) showing X-ray crystallographic evidence of double helical structure.

Ironically, Linus Pauling had published a proposed structure for nucleic acids – a triple helix – earlier in the year, because he did not have good X-ray crystallography data.

The details of the double helix• The two strands of DNA run anti-

parallel (Watson and Crick).• The two strands share a common

helix around which they wind; there is a major groove and a minor groove (Wilkins et al.).

• The bases are in the core of the structure and the phosphates make up the “backbone” of the helices (Franklin and Gosling).

• The bases form hydrogen bonds with complementary bases (“base pairing”) to connect two strands (Watson and Crick).

Double helix has a handedness

• DNA (both helices) is right-handed.

Base pairing

• Note that it is always a purine base pairing with a pyrimidine base.

• A/T pair is held together by two hydrogen bonds; G/C pair, three hydrogen bonds.

• Thus, one DNA strand can act as a template for building the other strand.

Describing DNA organization

• Genome chromosomes genes• Measure DNA length in terms of base pairs

(bp) or bases (b); a kilobase (kb) is a commonly used unit. 250000 kb in mammalian DNA.

• Each chromosome is a separate DNA molecule.

• Two sets of identical chromosome = diploid; one set = haploid.

RNA can form stem-loops

• RNA is single-stranded.

• Segments within RNA are self-complementary and can base pair with other segments to form “stem-loops”.

• Important structure in tRNA, for instance.

Brief history of DNA function

• Gregor Mendel (1866) – “factors” passed on by inheritance

• Fredrick Griffith (1928) – living nonvirulent pneumococci bacteria added to dead virulent pneumococci leads to living virulent bacteria. “transformation” by “Inheritance molecule”.

• Oswald Avery, Colin MacLeod and Maclyn McCarty (1944) – transformation of nonvirulent pneumococci to virulence by DNA transfer

Brief history of DNA function

• George Beadle and Edward Tatum (1941) found that genes lead to the production of enzymes. “One gene, one enzyme” theory.

• Francis Crick (1958) – “Central dogma” of biology (DNA RNA proteins)

• Sidney Brenner and Francis Crick (1961) – The codon is a three-nucleotide sequence in DNA that “codes” for a particular amino acid.

• Marshall Nirenberg (1961) – deciphers first codon (UUU codes for phenylalanine); in five years, he and coworkers decode the rest of the genetic code.

Basic ideas behind the central dogma

• DNA is replicated during cell division to make a copy of the DNA.

Basic ideas behind the central dogma• When the gene

is expressed, an messenger RNA (mRNA) copy is transcribed from the DNA.

• Finally, the mRNA is transported to the ribosome where it is translated into a peptide sequence using transfer RNAs (tRNA).

Detail of translation of proteins

Sequencing DNA• To determine the 5’ to 3’

sequence of the nucleotides in a DNA strand, one could hydrolyze the phosphodiester bond on either side and remove one nucleotide at a time and identify it.

• Robert Holley and others (“Nucleotide Sequences in the Yeast Alanine Transfer Ribonucleic Acid”, J. Biol. Chem. (1965)) took seven years to sequence a 76-nucleotide tRNA.

Faster way• Restriction

endonucleases exist in bacteria to resist the actions of bacteriophages.

• These enzymes cleave at a particular DNA sequence, typically 4 or 6 nucleotides long.

• Smith, Kelly and Wilcox (1970) isolate the first enzyme HindII, for which they share the Nobel Prize in 1978.

Restriction mapping• By overlapping

restriction “sites” on the target DNA, a length of DNA can be ordered by electrophoresis.

• Fragments thus cleaved can be “blunt-ended” or “sticky-ended”.

Gel electrophoresis

• DNA is negatively charged so it will be attracted to the anode of an electrical cell.

• Like protein gels, the migration distance is inversely proportional to the size (molecular weight) of the DNA fragment.

Chain terminator (dideoxy) method• The discovery of DNA polymerase I by Arthur Kornberg

(Washington University) in 1956 allowed Walter Gilbert (Harvard) and Frederick Sanger (Cambridge) twenty years later to develop a much quicker sequencing system.

Make every possible chain length

• Use DNA polymerase to make copies of the target DNA, use radioactively labeled dideoxy (ddNTP) to end the strand growth, then electrophoresis to separate different lengths.

Dideoxynucleotides (ddNTP)

• Terminate chain growth because no 3’ hydroxy group to attach a phosphate!

Just read it off!

• Automated at this point in time, can sequence kb in minutes.

Shotgun sequencing The limit of the Sanger method is < 1000 bp. By breaking up the original sequence into multiple random small fragments (<1000 bp), and doing the breakup several times, and sequencing all the fragments, overlapping sequences can be combined to yield the overall sequence.

Human genome project

• In 1990, the US DOE and NIH started a $3 billion project to map the 3.3 billion bases of the human genome.

• In 1998, a more modest private effort was started by Craig Venter and Celera Genomics.

• Between the two, the complete “final draft” human genomic sequence was published in 2003, a decade earlier than predicted.

• President Clinton signed an executive order that stated the human genome could not be patented; Celera stock plummets.

Human genome project results• 1. There are approximately 23,000 genes in human

beings, the same range as in mice and roundworms. • 2. Between 1.1% to 1.4% of the genome sequence codes

for proteins• 3. The human genome has significantly more duplicated

segments within it that other mammalian genomes do. These sections may be the source of new primate-specific genes.

• 4. At the time when the draft sequence was published less than 7% of protein families appeared to be vertebrate-specific.

• 5. Two randomly selected human genomes differ, on average, by only 1 nucleotide per 1250.

Cloning simply means adding a section of foreign DNA into a vector DNA (and having it copied)

• Typical vectors are plasmids, single-stranded circular DNA that replicate within bacteria.

• Plasmids can’t accept foreign DNA larger than 10 kb.

• Ligase is the enzyme used to repair open section of vector DNA.

How to tell if the foreign DNA has “taken”

• Disrupt a non-critical gene in the vector – for instance the beta-galactosidase gene that will result in the vector’s inability to cleave a galactose’s anomeric bond (and thus keep colonies of that form of the vector colorless, instead of turning blue).