分子生物学(英文版)

Chapter 3 Nucleic Acid

1. Physical and chemical structure of DNA

●Double-stranded helix

● Major groove and minor groove

● Base pairing

● The two strands are antiparallel

● G+C content (percent G+C)

● Satellite DNA

Satellite DNA consists of highly repetitive DNA and is so called because repetitions of a short DNA sequence tend to produce a different frequency of the nucleotides adenine, cytosine, guanine and thymine, and thus have a different density from bulk DNA - such that they form a second or 'satellite' band when genomic DNA is separated on a density gradient.

2. Alternate DNA structure

Two bases have been extruded from base stacking at the junction. The white line goes from phosphate to phosphate along the chain. O is shown red, N blue, P yellow and C grey.

3. Circular and superhelical DNA

DNA can also form a double-stranded, covalently-closed circle. These circular molecules are often coiled into a superhelix, the formation of which is catalyzed by enzymes called topoisomerases.

4. Denaturation of DNA

Denaturation: A transition from the native to the denatured state

DNA denaturation: also called DNA melting, is the process by which double-stranded DNA unwinds and separates into single-stranded strands through the breaking of hydrogen bonding between the bases.

Hyperchromicity/ Hyperchromic effect: the striking increase in absorbance of DNA (A260) caused by the denaturation of the double-stranded DNA molecule

Melting temperature (Tm) : the temperature at which half of the DNA strands are in the double-helical state and half are denatured. The melting temperature depends on both the length of the molecule, and the specific nucleotide sequence composition of that molecule.

Factors Affecting Tm

●G-C content of sample

● reagents that increase the solubility of the bases (anything that disrupts H-bonds or base stacking)

● Salt concentration

● pH

● Length

5. Renaturation

Strands can be induced to renature (anneal) under proper conditions. Factors to consider:

● Temperature

● Salt concentration

● DNA concentration

● Time

Repetitive Sequences

●Unique: Single Copy Genes

● Slightly repetitive (2-10 copies)

● Middle repetitive (10- hundreds)

--Clustered

--Dispersed

● Highly repetitive (hundreds to millions)

--Short sequences in satellite DNA

--Sequences of normal length in certain genes that exist in very large numbers

C-value Paradox

There is apparently a lack of association between C-value (the amount of DNA present in the haploid genome of different organisms )and the degree of organismal complexity of various

multi-cellular organisms. In 1971, Thomas named this phenomenon, “C-value Paradox”.

在每一种生物中其单倍体基因组的DNA总量是特异的，被称为C值 (C Value)。 C值和生物结构或组成的复杂性不一致的现象称为C值悖论(C-value paradox)。

6. Hybridization

Hybridization: the technique wherein renatured DNA is formed from separate single-stranded samples .

Heteroduplexing: renaturation combined with electron microscopy in a procedure allows the localization of common, distinct,and missing sequences in DNA.

DNA-RNA hybridization (Northern hybridization): the use of filter hybridization to detect sequence complementarity between a single strand of DNA and an RNA molecule.

7. The structure of RNA

Types: mRNA, tRNA, rRNA

Distinctions:

- ribose replaces deoxyribose;

- U replaces T;

- Single-stranded

Conformation: stem-loop or hairpin

8. Hydrolysis of nucleic acid

The phosphodiester bonds of both DNA and RNA can be broken by hydrolysis either chemically or enzymatically.

Ribozymes: the RNA enzymes, are able to cleave and form specific phosphodiester bonds in a manner analogous to protein enzymes.

Chapter 6 The genetic material

The Path to the Watson and Crick Model

1928, Griffith, transformation in pneumococci（肺炎球菌）

1944, Avery, Griffith’s transforming principle was DNA

1950, Chargaff, a pattern in the amounts of the four bases

1952, Hershey and Chase, DNA is the genetic material

1953, Franklin, the x-ray picture of DNA

Chargaff’s rule

In the DNA of all species examined, A=T, G=C

The total amount of purines (A+G)=pyrimidines (T+C) in DNA

The ration of (A+T)/(G+C) varies from species to species

DNA properties and functions

1.DNA has the ability to store genetic information, which can be expressed in the cell as need.

2.This information can be transmitted to daughter cells with minimal error. (This process requires complex enzymes and repair mechanisms.)

3.DNA possesses both physical and chemical stability so information is not lost over long periods of time (years).

4.DNA has the potential for heritable change without major loss of parental information. DNA-genetic material: Double-stranded DNA has evolved as the genetic material because it is especially well-suited for replication, repair, occasional change, and long-time stability. Gene: Genes contain all the information for the synthesis and functioning of cellular components. Transcription: the process of synthesizing RNA molecules from a DNA template.

Triplets / codons: the RNA nucleotide sequence is read (on ribosomes) in sequential groups of three bases.

Mutation: the process by which a base-sequence changes.

The central dogma: DNA makes RNA, makes protein.

chapter 7 DNA replication

Semiconservative replication of double-stranded DNA

Untwisting of highly coiled DNA is required for DNA replication

Topoisomerase Type I :

?Work ahead of replicating DNA

?Mechanism

–Makes a cut in one strand, passes other strand through it. Seals gap.

–Result: the DNA is “relaxed” somewhat

Gyrase--A Type II Topoisomerase

–Introduces negative supercoils

–breaks both strands

–Section located away from actual cut is then passed through cut site.

Initiation of DNA replication

?Replicaion initiated at specific sites: Origin of Replication (ori)

?Two Types of initiation:

–De novo –Synthesis initiated with RNA primers. Most common.

–Covalent extension—synthesis of new strand as an extension of an old strand (“Rolling Circle”). Limited to certain viruses.

De novo Initiation

?Binding to Ori C by DnaA protein

?Opens Strands

?Replication proceeds bidirectionally

Covalent extension initiation Rolling Circle

Unwinding of DNA for replication

Helicase:

? Breaks hydrogen bonds and eliminates hydrophobic interactions

? Needs energy supplied by ATP

? Encoded by the DnaB gene in E.coli

Single-strand DNA binding proteins (SSB):

Bind to the exposed strands, coat them and block the re-annealing process.

Elongation of newly synthesized strands

1.The polymerization reaction and the polymerases

Enzyme: polymerase III

Needed: substrates, template, primer

Direction: 5’→3’

2. Correcting mismatched bases

The 5’-3’ exonuclease activity of pol I at a single-strand break (nick) can occur simultaneously

with polymerization----nick translation.

DNA polymerase III consists of multiple subunits

?Pol I and pol III are both involved in E.coli DNA replication. Pol III is the major

polymerase.

? Both poly I and poly III possess a proofreading or editing function (3’-5’ exonuclease

activity ).

? The 5’-3’ exonuclease activity of pol I at a single-strand break (nick) can occur

simultaneously with polymerization----nick translation.

? DNA polymerase III consists of multiple subunits.

? All known polymerases can work only in the 5’-P → 3’-OH direction.

Pol I and pol III have some features in common:

● 5’-3’ polymerization activity

The four deoxynucleoside 5’-triphosphates

A primer with a free 3’-OH

A template

● 3’-5’ exonuclease activity

Antiparallel DNA strands and discontinuous replication

?The two strands of DNA is antiparallel and the replication is discontinuous synthesis.

? A primer is required for chain initiation and two different enzymes (RNA polymerase and

primase) are known to synthesize primer RNA molecules.

? DNA ligase joins precursor fragments and pol I as well as RNase H participates in the

removal of primer.

RNA polymerase: initiation of leading-strand synthesis

Primase: synthesis of primers for lagging-strand

Primosome: helicase/primase complex

Pol I: removal of the primer and replacement of DNA

DNA ligase: joining the fragment (gap sealed)

The complete DNA replication system

Bidirectional replication speeds up DNA synthesis

Replication of eukaryotic chromosomes

1.Eukaryotes have more and large chromosomes.

2.Eukaryotic replication may require as much as 6-8 hours for completion versus the 40

minutes needed by E.coli.

3.There are multiple, rather than a single, replication origins along eukaryotic chromosomes.

They are spaced about 20 kb apart.

4.Eukaryotic DNA replication is at the rate of about 10-100 nucleotides per second as opposed

to the prokaryotic rate of about 1500 nucleotides per second.

5.At least five types of DNA polymerases have been found in eukaryotic cells.

真生物DNA的复制有DNA聚合酶及多种蛋白质因子参与，DNA聚合酶也有多种类型。其中DNA Polα

及DNA Polδ在细胞核内DNA的复制中起主要作用。DNA Polδ催化前导链及滞后链的合成，是主要负责DNA复制的酶。DNA Polα的功能主要是引物合成。DNA Polγ是线粒体中的复制酶。

Chapter 8 Transcription

1. Enzymatic synthesis of RNA

E. Coli RNA polymerase

Holoenzyme:

core enzyme: α2ββ’ω

σfactor

(1)Binding of RNA pol to a template at specific site

(2) Initiation

(3) Chain elongation

(4) Chain termination and release

2. Transcription signals

In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site.

●the -10 element :Pribnow box, usually consists TATAAT, is absolutely essential to start

transcription in prokaryotes.

●the -35 element:usually consists of TTGACA. Its presence allows a very high transcription

rate.

In prokaryotes:

In eukaryotes:

Termination

Termination of RNA synthesis occurs at specific base-sequences in the DNA molecule, called terminators.

?Intrinsic terminators:rho-independent terminators, the termination sequences allow RNA polymerase to terminate elongation spontaneously.

? rho-dependent terminators: it is dependent on a specific protein called a rho factor. Intrinsic Termination

?RNA pol passes over inverted repeats

?Hairpins begin to form in the transcript

?Poly-U:poly-A stretch melts

? RNA pol and transcript fall off

Rho: Mechanism

?Rho binds to transcript at loading site (up stream of terminator)

?Hairpin forms, pol stalls

?Rho helicase releases transcript and causes termination

3. Classes of RNA molecules

Messenger RNA: short lifetime

Ribosomal RNA

Transfer RNA

cistron: a DNA segment corresponding to one polypeptide chain plus the start and stop signals monocistronic mRNA: an mRNA encoding a single polypeptide

polycistronic mRNA: an mRNA encoding several different polypeptide chains

RNA processing is to generate a mature mRNA (for protein genes) or a functional tRNA or rRNA from the primary transcript.

Processing of pre-mRNA involves the following steps:

Capping: add 7-methylguanylate (m7G) to the 5' end.

Polyadenylation: add a poly-A tail to the 3' end.

Splicing: remove introns and join exons.

In some cases, RNA editing is also involved.

Processing of pre-rRNA and pre-tRNA:

The newly transcribed pre-rRNA is a cluster of three rRNAs: 18S, 5.8S and 28S in mammals. They must be separated to become functional. Pre-rRNA is synthesized in the nucleolus（核仁）. The