NUCLEINUCLEIC C

ACIDSACIDS(2)(2)

OBJECTIVES OBJECTIVES Denaturation of DNA Denaturation of DNA Identify/ recognize RNAIdentify/ recognize RNA Differentiate between DNA and RNADifferentiate between DNA and RNA Differentiate between mRNA, tRNA, rRNADifferentiate between mRNA, tRNA, rRNA

DNADNA Can be disrupted by Can be disrupted by heat, acids, bases or heat, acids, bases or organic solvents organic solvents ((double helix double helix denatured = unwinding denatured = unwinding of the DNA double of the DNA double helixhelix))

In nature, the In nature, the unwinding of the DNA unwinding of the DNA double helix is the double helix is the important step in DNA important step in DNA replicationreplication Involves the nitrogenous bases

Denaturation of DNADenaturation of DNA Denaturation:Denaturation: disruption of 2° disruption of 2°

structurestructure– most commonly by heat most commonly by heat

denaturation (denaturation (melting- melting- the heat the heat denaturation of DNA)denaturation of DNA)

– as strands separate, absorbance as strands separate, absorbance at 260 nm increasesat 260 nm increases

– increase is called hyperchromicity-increase is called hyperchromicity-the wavelength of absorption does not change but the wavelength of absorption does not change but the amount of light absorbed increasesthe amount of light absorbed increases

– midpoint of transition (melting) midpoint of transition (melting) curve = Tcurve = Tmm

– the the higher the % G-C, the higher higher the % G-C, the higher the Tthe Tmm

– Renaturation/annealing Renaturation/annealing is possible is possible on on slow coolingslow cooling

Principal use in PCRPrincipal use in PCR

At the nitrogenous bases

Denaturation of DNADenaturation of DNA Double helix unwinds when DNA is denaturedDouble helix unwinds when DNA is denatured

Can be re-formed with slow cooling and annealingCan be re-formed with slow cooling and annealing

RNARNA consist of long, unbranched chains of nucleotides consist of long, unbranched chains of nucleotides

joined joined by phosphodiester bonds between the 3’-OH by phosphodiester bonds between the 3’-OH of one pentose and the 5’-P of the next nucleotideof one pentose and the 5’-P of the next nucleotide

the the pentose unit is pentose unit is -D-ribose-D-ribose (it is 2-deoxy-D- (it is 2-deoxy-D-ribose in DNA)- the extra OH present in RNA makes ribose in DNA)- the extra OH present in RNA makes this nucleotide more susceptible to hydrolysis than this nucleotide more susceptible to hydrolysis than DNA.DNA.

the pyrimidine bases are the pyrimidine bases are uraciluracil and cytosine (they and cytosine (they are are thyminethymine and cytosine in DNA) and cytosine in DNA)

RNA is single stranded (DNA is double stranded)RNA is single stranded (DNA is double stranded)

The bases sequences of all types of RNA are determined by that of DNA

RNARNA RNA molecules are classified according to RNA molecules are classified according to

their structure and functiontheir structure and function

RNA structureRNA structure Levels of structureLevels of structure

– 1° structure: the order of bases on 1° structure: the order of bases on the polynucleotide sequence; the polynucleotide sequence; complementary to the DNA templatecomplementary to the DNA template

– 2° structure: no specific 2° 2° structure: no specific 2° arrangements, but RNA is not arrangements, but RNA is not completely lacking of regular completely lacking of regular structurestructure

– 3° structure:interaction between DNA 3° structure:interaction between DNA and proteins and proteins

RNA- 1° StructureRNA- 1° Structure Polymer of nucleotidePolymer of nucleotide Involves single polynucleotide strandInvolves single polynucleotide strand

RNA- 2° StructureRNA- 2° Structure Loop back onto themselves to Loop back onto themselves to

fold into conformation containing fold into conformation containing several different structural several different structural elements:elements:

1-hairpin turns1-hairpin turns

2-right-handed double helixes2-right-handed double helixes

3-internal loops3-internal loops

All classes of RNA synthesized All classes of RNA synthesized as single-stranded moleculesas single-stranded molecules

3’OH 5’P

Hairpin turnHairpin turn- Loops in the single chainLoops in the single chain

Right-handed double helixesRight-handed double helixes- result of - result of intrastrandintrastrand foldingfolding- Trigger by hairpin turnTrigger by hairpin turn- Antiparallel & stabilized in the same Antiparallel & stabilized in the same

direction as in DNAdirection as in DNA- Hold by H bond & stacking interactionHold by H bond & stacking interaction

Internal loopsInternal loops- Common in RNACommon in RNA- Structural features that disrupt the Structural features that disrupt the

formation of continuous double helix formation of continuous double helix regionsregions

tRNAtRNA Transfer RNA, Transfer RNA,

tRNA: tRNA: – the smallest kind of the smallest kind of

the three RNAsthe three RNAs– a a single-stranded single-stranded

polynucleotide chainpolynucleotide chain between 73-94 between 73-94 nucleotide residuesnucleotide residues

– carriescarries an an amino amino acid at its 3’ endacid at its 3’ end

– intramolecular intramolecular hydrogen bondinghydrogen bonding occurs in tRNAoccurs in tRNA

Cloverleaf structure

Function: Involves in synthesis of polypeptide, to carry amino acid to site of protein synthesis

tRNA structuretRNA structure Smallest types of RNASmallest types of RNA Highly structured Highly structured All tRNAs contain between 74 and 93 nucleotides in All tRNAs contain between 74 and 93 nucleotides in

a single chaina single chain Structural features: hairpin turns, regions of double Structural features: hairpin turns, regions of double

helix and loops (non-hydrogen bonded portions) helix and loops (non-hydrogen bonded portions) Carriers of specific amino acids used for protein Carriers of specific amino acids used for protein

synthesissynthesis Reads the codon message on mRNA and Reads the codon message on mRNA and

incorporates amino acid into the protein being incorporates amino acid into the protein being synthesizedsynthesized

20 amino acid – 20 tRNA20 amino acid – 20 tRNA

tRNA – 3tRNA – 3oo structure structure To produce tertiary structure, tRNA folds into an L-To produce tertiary structure, tRNA folds into an L-

shaped conformation shaped conformation

The 3D structure of yeast tRNA The 3D structure of yeast tRNA for phenylalaninefor phenylalanine

rRNArRNA Ribosomal RNA, rRNA:Ribosomal RNA, rRNA: a ribonucleic acid a ribonucleic acid

found in ribosomes, the site of protein found in ribosomes, the site of protein synthesissynthesis– only a few types of rRNA exist in cellsonly a few types of rRNA exist in cells– ribosomes (ribosomes (protein-synthesizing organelles) protein-synthesizing organelles)

consist of 60 to 65% rRNA and 35 to 40% proteinconsist of 60 to 65% rRNA and 35 to 40% protein– in both prokaryotes and eukaryotes, ribosomes in both prokaryotes and eukaryotes, ribosomes

consist of two subunits, one larger than the otherconsist of two subunits, one larger than the other– analyzed by analytical ultracentrifugationanalyzed by analytical ultracentrifugation– particles characterized by sedimentation particles characterized by sedimentation

coefficients, expressed in Svedberg units (S)coefficients, expressed in Svedberg units (S)– SequencingSequencing of of 16S RNA (small subunit of 16S RNA (small subunit of

bacteria rRNA) - identification of bacteriabacteria rRNA) - identification of bacteria

rRNA structurerRNA structure Secondary & tertiary structures of rRNA Secondary & tertiary structures of rRNA

display same elements as tRNAsdisplay same elements as tRNAs

Secondary structure for Secondary structure for E. coliE. coli 16S rRNA. 16S rRNA.

mRNAmRNA Messenger RNA, mRNA:Messenger RNA, mRNA:

a ribonucleic acid that carries coded a ribonucleic acid that carries coded genetic information from DNA to genetic information from DNA to ribosomes for the synthesis of proteinsribosomes for the synthesis of proteins– present in cells in relatively small amounts and present in cells in relatively small amounts and

very short-lived (less abundant form of RNA) very short-lived (less abundant form of RNA) – single strandedsingle stranded– biosynthesis is directed by information biosynthesis is directed by information

encoded on DNAencoded on DNA– Synthesize from DNA, the nucleotide sequence Synthesize from DNA, the nucleotide sequence

in mRNA is similar with the 5’-3’ strand of DNA, in mRNA is similar with the 5’-3’ strand of DNA, with the exception of U replacing Twith the exception of U replacing T

5’-3’ DNA sequence is the same with RNA sequence (complementary to 3’-5’DNA) sequence

Structure: Linear polynucleotide strand

mRNA structuremRNA structure Serves as a template for protein synthesis (Carries Serves as a template for protein synthesis (Carries

the transient message for protein synthesis from the transient message for protein synthesis from nuclear DNA to the ribosomes)nuclear DNA to the ribosomes)

Move the information contained in DNA to the Move the information contained in DNA to the translation machinerytranslation machinery

Each molecule carries the instruction for each gene Each molecule carries the instruction for each gene (codes for one type of polypeptide product)(codes for one type of polypeptide product)

5’ – G G C A U U G C G C - 3’5’ – G G C A U U G C G C - 3’

Initiation codon Initiation codon - codes for the 1codes for the 1stst amino acid in all polypeptide amino acid in all polypeptide

sequences (AUG)sequences (AUG)- N-formyl methionine in prokaryotes and N-formyl methionine in prokaryotes and

methionine in eukaryotesmethionine in eukaryotes

Termination codon Termination codon - UAA, UAG & UGAUAA, UAG & UGA- do not code for an amino acid & thus signal do not code for an amino acid & thus signal

the end of protein synthesisthe end of protein synthesis- Also called stop codon or nonsense codonAlso called stop codon or nonsense codon