Chapter 2 DNA, RNA, Transcription and Translation

I. DNA (deoxyribonucleic acid) 

The basic genetic material to establish and maintain the cellular and biochemical function.

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Central Dogma: (Gene Expression)

Structure of DNA (discovered by Watson and Crick) 

DNA basic unit: nucleotides that are composed of an organic base, a pentose and a phosphate group.

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Four different bases in DNA (A, T, G, C):

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The genetic information is stored in the alignment and sequence of these 4 bases, analogous to 0 and 1 used in the information storage in computer. The sequence of the DNA determines the polypeptide sequence and the protein function and hence the cellular activities and functions.

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Precursor of DNA synthesis: (deoxynucleoside triphosphate)

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The nucleotides of DNA are joined by the addition of dNTP to the polynucleotide chain (the P at the  position reacts with the 3’ –OH at the growing nucleotide chain. **DNA synthesis proceeds from 5’ to 3’ ＝＞ 5’－ATGC－….3’

Base

nucleoside

nucleotide

Precursors for

Precursors for

DNA synthesis

RNA synthesis

Adenine (A)

Adenosine

Adenylic acid

dATP

ATP

Guanine (G)

Guanosine

Guanylic acid

dGTP

GTP

Cytosine (C)

Cytidine

Cytidylic acid

dCTP

CTP

Thymine (T)

Thymidine

Thymidylic acid

dTTP

Uracil (U)

Uridine

Uridylic acid

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Note: 

base + sugar = nucleoside

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phosphate + base + sugar = nucleotide

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UTP

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ATP: adenosine triphosphate, energy stored in ATP can drive many bioreactions (e.g. active transport by hydrolyzing to ADP or AMP).

DNA double helix 

DNA in cells exists as a double helix, consisting of two long chains (strands).

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A only pairs with T, G only pairs with C. These reactions are called base pairing, the two strands are complementary. The length of DNA is expressed in base pair (bp).

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The two strands run in opposite direction ＝＞ one is 5’→3’, the other is 3’→5’ ＝ ＞ because both strands are complementary ∴ if one strand is 5’－ TAGGCAT－3’ the other strand must be 3’－ATCCGTA－5’

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Usually 5’ end starts from the left.

II. DNA Replication [5]: 

During the cell division, new DNA is synthesized and segregated to new daughter cells. The replication initiates at origin of replication (ori).

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Some ori are identified in bacteria (245 bp oriC in E. coli), yeast, chloroplasts and mitochondria. ori is usually A-T rich (easier melting).

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DNA topoisomerase: unwinds the helix

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DNA helicase: unzip the helix

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DNA polymerase: moves along the DNA template and catalyzes the incorporation of nucleotides into DNA (synthesis from 5’ to 3’). Replication process in E. coli (500 nt/s in bacteria, 50 nt/s in mammals)

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Synthesis of lagging strand. See Ref [2]

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DNA polymerase requires an RNA primer to initiate DNA synthesis, but RNA polymerase doesn’t need an RNA primer for RNA synthesis.

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In eucaryotes, repair system exists to ensure the fidelity of DNA replication.

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In E. coli, chromosome is circular, replication moves toward two directions1.

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In each daughter chromosome, one strand comes from the parent, the other is newly synthesized, ∴ → semiconserved synthesis.

Gene: 

A stretch of nucleotide bases on a stand of DNA that is transcribed into RNA.

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In human cells, the telomere is synthesized by telomerase. Telomerase is absent in many cell types thus their telomeres become

shorter with each cell division eventually DNA damage occurs at chromosome ends sends a signal to stabilize p53transcription of several genes (e.g. p21) CKI (cdk inhibitor protein) bind to and inhibit G1/S-cdk and S-cdk.=> block entry into S phase (Alberts, p10071018).

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The number and sequence of the bases within a gene determine the info the gene carries －i.e. the a.a. sequence of the specific polypeptide.

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There are

III. RNA (ribonucleic acid): 

RNA is also a polymer of nucleotides, but is different from DNA in that 

U (uracil) substitutes for thymine (T) in RNA.

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The pentose is ribose, instead of deoxyribose.

Types of RNA 

mRNA (messenger RNA): encodes protein (3~5%); transcribed by RNA polymerase II (RNA pol II). 

DNA is always present within the cell, whereas mRNA is present transiently, degraded after a short period of time (Half-lives of yeast mRNA:1-60 min. Halflives of animal mRNA:1-24 h).

 

DNA is double stranded, whereas mRNA is single stranded.

tRNA (transfer RNA): carries a.a to the site of protein synthesis (required for protein translation) (4%); transcribed by RNA pol III.

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rRNA (ribosomal RNA): several different sizes (≒90%); transcribed by RNA pol I. 

5S, 16S, 23S in procaryotes. (S is the relative sedimentation rate during centrifugation)

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5S, 5.8S, 18S, 28S in eucaryotes, extensively modified (e.g. methylation of the 2’OH position on ribose)

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rRNA combines with some proteins to form ribosome microRNA:

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Catalytic RNA (ribozyme): enzymatically cleave RNA molecules.

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IV.  

Transcription [2, 3, 5]: RNA polymerase (RNA pol) is the enzyme to catalyze the transcription using DNA as the template (40 nt/s at 37C for bacterial RNA pol, much slower than the DNA replication rate). RNA pol first binds the promoter region of the template DNA. RNA pol is large and spans 75-80 bp (from -55 to +20), it separates the two DNA strands in a transient bubble and synthesizes the first 9 nt bond.

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RNA pol moves along the template strand from 3’ to 5’ direction in a way similar to DNA replication (RNA synthesis must be from 5’ to 3’).

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As RNA pol moves, it unwinds the DNA at the front of the bubble (12-14 bp) and rewinds the DNA at the back. The RNA-DNA hybrid is shorter and transient, then the nascent RNA is released.

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When the termination sequence is reached, the enzyme stops adding nt to the RNA chain, releases the product and dissociates from the DNA template.

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Note: 

RNA pol consists of the following subunits 

2  subunits: enzyme assembly and promoter recognition

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 and ’ subunits: catalytic center

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 subunit: promoter specificity. In E. coli, the  factor (e.g. 70, 32) is essential for initiation, it’s released when RNA chain reaches 8-9 nt.

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Two strands of DNA 

coding strand (sense strand):

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template strand (antisense strand):

Rifampicin, an antibiotic used against tuberculosis, inhibits transcription.

Differences in procaryotic and eucaryotic genes:

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Procaryotic genes 

Genes w/ related functions are often contiguous, forming the operon (including the genes themselves and the control element). These genes are under the control of a single promotergenerates a set of proteins (polycistronic).

Ex: Lac Operon (1st operon studied and uncovered by Jacob and Monod in 1961), the gene products enable cells to take up and metabolize -galactoside such as lactose. 

LacZ: encodes -galactosidase, cleaves lactose into glucose and galactose

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LacY: encodes permease, transports -galactosides2 into the cell.

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LacA: encodes transacetylase, transfers an acetyl group from acetyl-CoA to galactosides

Note: the operon maintains basal level transcription so that small amounts of permease can transport foreign -galactosides into the cells.

Eucaryotic genes: 

Consist of a set of coding regions (exon) separated by noncoding regions (intron). mRNA is synthesized via transcription and undergoes splicing.

2

Galactoside: a glycoside (a molecule in which a sugar is bound to another functional group via a glycosidic bond) containing galactose.

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See Ref [4] 

All RNA pol II transcribed RNAs are capped by a terminal nt, 7methylguanylate (m7G). The 5’ cap positively influences the poly A addition and splicing, and is essential for the initiation of translation.

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After transcription, the 3’ end of mRNA is cleaved by an

See Ref [1]

endonuclease, then poly(A) polymerase adds 200 A residues downstream (15 nt) of the polyA signal (e.g. AAUAAA) 

Splicing occurs in spliceosome (in the nucleus) which consists of small nuclear RNA (