nucleic acid
DNA function
The main genetic material is the carrier of genetic information
RNA function
Participate in protein biosynthesis
In RNA viruses, RNA is the genetic material
RNA post-transcriptional processing and modification
Involved in the regulation of gene expression and cell function
Nucleotide
base
Cytosine (C), Uracil (U), Thymine (T)
Rare nucleotides: such as IMP (important substance for nucleic acid metabolism)
pentose sugar
Pentose sugar in DNA: β-D-2′-deoxyribose
Pentose sugar of RNA: β-D-ribose
nucleoside or deoxynucleoside
DNA
primary structure
Refers to the connection mode and arrangement order between deoxynucleotides. The connection mode is 3′, 5′-phosphodiester bond.
secondary structure
Double helix structure (proposed by Watson and Crick)
Antiparallel right-handed double helix with major and minor grooves on the surface
The deoxyribose-phosphate backbone is outside and the complementary base pairing is inside
The helix diameter is 2nm, the vertical distance between adjacent base planes is 0.34nm, each turn is 10bp, and the helix pitch is 3.4nm.
The main forces that stabilize the double helix structure: base stacking forces and base pairing hydrogen bonds
DNA base composition law (Chargaff law)
Law of base equivalence: In the DNA of almost all organisms, A=T, G=C, A G=T C
DNA base composition is species-specific, and the asymmetry ratio (A T)/(G C) varies from species to species
tertiary structure
Superhelical structure
Natural DNA mainly exists in a negative superhelical structure
RNA
mRNA
mRNA is the template for protein synthesis and mainly uses primary structure (ribonucleotide connection method and arrangement order) to transmit genetic information.
Molecular structure of prokaryotic mRNA
Composed of a leader region, several translation regions (cistrons) and terminal sequences
The 5’-end leader region has a sequence rich in purine bases, which is the SD sequence.
Molecular structure of eukaryotic mRNA
There is a cap structure at the 5' end, followed by the 5' untranslated region, the coding region, and the 3' untranslated region.
The 3' end is a poly(A) tail.
tRAN
Carry and transport activated amino acids, recognize the codons on the mRNA, and transport specific amino acids to the ribosome for protein synthesis according to the sequence of the genetic code on the mRNA.
Primary structure characteristics: Contains more rare nucleosides; 3′ end has CCA sequence
4 and 5 ´ ends are mostly pG (some are pC)
Secondary structure: clover-shaped structure, composed of four arms and four rings: dihydrouracil ring (D ring), D arm, anticodon ring, anticodon arm (AC arm), additional ring, TψC ring, TψC arm , amino acid arm (the 3´-CCA end is the binding site for activated amino acids)
Tertiary structure: inverted L-shaped structure
rRNA
Prokaryotic cells have 3 types of rRNA (5S, 16S, 23S rRNA)
There are 4 types of rRNA in eukaryotic cells (5S, 5.8S, 18S, 28S rRNA)
Function: The transferase activity that makes up ribosomes and catalyzes the formation of peptide bonds exists on 23S rRNA and is involved in the binding of tRNA and mRNA.
ribosome
Virus
non-cellular organisms composed of nucleic acids and proteins
chromosome
The basic structural unit of chromosomes and chromatin is the nucleosome, which is composed of DNA wrapped around histones.
Important physical and chemical properties
General properties:
(1) Crystal form: DNA in white fiber form, RNA in white powder form
(2) Solubility: soluble in water, insoluble in organic solvents
(3) Acidity and alkalinity: obvious acidity, isoelectric point as low as 2.0--2.5,
(4) Viscosity: DNA has high viscosity, RNA has low viscosity
(5) Amphoteric dissociation characteristics: alkaline hydrolysis and acid hydrolysis
Alkaline hydrolysis and acid hydrolysis characteristics:
(1) Under mild alkaline conditions: the phosphodiester bonds of DNA are stable, and the phosphodiester bonds of RNA are all decomposed into 2’ and 3’ cyclic nucleotides
(2) Exposing dilute acid for a long time (or increasing the temperature or acidity): Purine is separated and a small number of phosphodiester bonds are decomposed.
(3) Medium-strong acid or concentrated acid treatment: pyrimidine decomposes, and more phosphodiester bonds decompose
UV absorption characteristics: maximum absorbance near 260nm
Denaturation, renaturation and molecular hybridization of nucleic acids
Denaturation: Under the influence of physical and chemical factors, the hydrogen bonds between DNA base pairs are broken and the double helix is unraveled. This is a climacteric process, accompanied by an increase in A260 (hyperchromic effect) and loss of DNA function.
Denaturation factors: thermal denaturation, acid-base denaturation (pH less than 4 or greater than 11),
Denaturant (urea, guanidine hydrochloride, formaldehyde, etc.)
Tm: The temperature at which A260 reaches half of its maximum value during thermal denaturation is called the melting temperature of the DNA, represented by Tm
Renaturation: Under certain conditions, the bases between the denatured DNA single strands are re-paired to restore the double helix structure. Accompanied by a decrease in A260 (hypochromic effect), DNA function is restored.
Main factors affecting regeneration
1. Temperature: Thermal denatured DNA can be renatured when cooled slowly, but cannot be renatured by rapid cooling.
2. DNA concentration: The greater the concentration, the faster the renaturation
3. DNA fragment size: The larger the fragment, the slower the renaturation
4. The number of repeated sequences in a DNA fragment
5. Ionic strength of solution
Molecular hybridization: As long as there is a base pairing region between DNA single strands from different sources or between single stranded DNA and RNA, a local double helix region can be formed during renaturation, which is called nucleic acid molecular hybridization.
application:
1. Identification of purity (A280 is the maximum absorbance of proteins and phenolic substances)
The A260/A280 of pure DNA should be 1.8 (1.65-1.85)
The A260/A280 of pure RNA should be 2.0.
If it contains protein or phenols, the A260/A280 ratio will be significantly reduced.
2. Determine whether DNA is denatured
During the denaturation process of DNA, the molar absorption coefficient increases (chromic effect)
During the renaturation process of DNA, the molar absorption coefficient decreases (subtractive effect)
Among the three types of RNA, tRNA is the most abundant, followed by rRNA and very little mRNA.
Important experimental basis
① Pneumococcus transformation experiment
② Bacteriophage infection experiment
[Objective] Master the composition of nucleotides, Chargaff's law, the structural characteristics and functions of DNA, tRNA, rRNA and mRNA, especially the key points and significance of the DNA double helix structure model. Master the UV absorption properties, denaturation and renaturation of nucleic acids, and molecular hybridization.
[Key points] The structure of DNA, the structural characteristics and functions of tRNA, mRNA, and rRNA, and the physical and chemical properties of nucleic acids.
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