1. NUCLEIC ACIDS (2)

2. OBJECTIVES Denaturation of DNA Identify/ recognize RNA
Differentiate between DNA and RNA
Differentiate between mRNA, tRNA, rRNA

3. DNA
Can be disrupted by heat, acids, bases or organic solvents (double helix denatured = unwinding of the DNA double helix)
In nature, the unwinding of the DNA double helix is the important step in DNA replication
Involves the nitrogenous bases 3

4. Denaturation of DNA Denaturation: disruption of 2° structure
most commonly by heat denaturation (melting- the heat denaturation of DNA)
as strands separate, absorbance
at 260 nm increases
increase is called hyperchromicity-the wavelength of absorption does not change but the amount of light absorbed increases
midpoint of transition (melting) curve = Tm
the higher the % G-C, the higher the Tm
Renaturation/annealing is possible on slow cooling
Principal use in PCR
At the nitrogenous bases 4

5. Denaturation of DNA Double helix unwinds when DNA is denatured
Can be re-formed with slow cooling and annealing 5

6. The bases sequences of all types of RNA are determined by that of DNA
consist of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3’-OH of one pentose and the 5’-P of the next nucleotide
the pentose unit is -D-ribose (it is 2-deoxy-D-ribose in DNA)- the extra OH present in RNA makes this nucleotide more susceptible to hydrolysis than DNA.
the pyrimidine bases are uracil and cytosine (they are thymine and cytosine in DNA)
RNA is single stranded (DNA is double stranded)
The bases sequences of all types of RNA are determined by that of DNA 6

7. RNA
RNA molecules are classified according to their structure and function 7

8. RNA structure Levels of structure
1° structure: the order of bases on the polynucleotide sequence; complementary to the DNA template
2° structure: no specific 2° arrangements, but RNA is not completely lacking of regular structure
3° structure:interaction between DNA and proteins 8

9. RNA- 1° Structure Polymer of nucleotide
Involves single polynucleotide strand

10. All classes of RNA synthesized as single-stranded molecules
RNA- 2° Structure
Loop back onto themselves to fold into conformation containing several different structural elements:
1-hairpin turns
2-right-handed double helixes
3-internal loops
All classes of RNA synthesized as single-stranded molecules
5’P
3’OH

11. Hairpin turn
Loops in the single chain
Right-handed double helixes
- result of intrastrand folding
Trigger by hairpin turn
Antiparallel & stabilized in the same
direction as in DNA
Hold by H bond & stacking interaction
Internal loops
Common in RNA
Structural features that disrupt the
formation of continuous double helix regions

12. tRNA Transfer RNA, tRNA: the smallest kind of the three RNAs
Function: Involves in synthesis of polypeptide, to carry amino acid to site of protein synthesis
Transfer RNA, tRNA:
the smallest kind of the three RNAs
a single-stranded polynucleotide chain between nucleotide residues
carries an amino acid at its 3’ end
intramolecular hydrogen bonding occurs in tRNA
Cloverleaf structure 12

13. tRNA structure Smallest types of RNA Highly structured
All tRNAs contain between 74 and 93 nucleotides in a single chain
Structural features: hairpin turns, regions of double helix and loops (non-hydrogen bonded portions)
Carriers of specific amino acids used for protein synthesis
Reads the codon message on mRNA and incorporates amino acid into the protein being synthesized
20 amino acid – 20 tRNA

14. The 3D structure of yeast tRNA for phenylalanine
tRNA – 3o structure
To produce tertiary structure, tRNA folds into an L-shaped conformation
The 3D structure of yeast tRNA for phenylalanine

15. rRNA
Ribosomal RNA, rRNA: a ribonucleic acid found in ribosomes, the site of protein synthesis
only a few types of rRNA exist in cells
ribosomes (protein-synthesizing organelles) consist of 60 to 65% rRNA and 35 to 40% protein
in both prokaryotes and eukaryotes, ribosomes consist of two subunits, one larger than the other
analyzed by analytical ultracentrifugation
particles characterized by sedimentation coefficients, expressed in Svedberg units (S)
Sequencing of 16S RNA (small subunit of bacteria rRNA) - identification of bacteria 15

16. rRNA structure
Secondary & tertiary structures of rRNA display same elements as tRNAs
Secondary structure for E. coli 16S rRNA.

17. mRNA Messenger RNA, mRNA:
Structure: Linear polynucleotide strand
Messenger RNA, mRNA:
a ribonucleic acid that carries coded genetic information from DNA to ribosomes for the synthesis of proteins
present in cells in relatively small amounts and very short-lived (less abundant form of RNA)
single stranded
biosynthesis is directed by information encoded on DNA
Synthesize from DNA, the nucleotide sequence in mRNA is similar with the 5’-3’ strand of DNA, with the exception of U replacing T
5’-3’ DNA sequence is the same with RNA sequence (complementary to 3’-5’DNA) sequence 17

18. mRNA structure 5’ – G G C A U U G C G C - 3’
Serves as a template for protein synthesis (Carries the transient message for protein synthesis from nuclear DNA to the ribosomes)
Move the information contained in DNA to the translation machinery
Each molecule carries the instruction for each gene (codes for one type of polypeptide product)
5’ – G G C A U U G C G C - 3’

19. Initiation codon
codes for the 1st amino acid in all polypeptide sequences (AUG)
N-formyl methionine in prokaryotes and methionine in eukaryotes
Termination codon
UAA, UAG & UGA
do not code for an amino acid & thus signal the end of protein synthesis
Also called stop codon or nonsense codon