Download Chapter 2 DNA, RNA, Transcription and Translation I. DNA

Chapter 2 DNA, RNA, Transcription and Translation
I. DNA (deoxyribonucleic acid)

The basic genetic material to establish and maintain the cellular and biochemical
function.

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.

Four different bases in DNA (A, T, G, C):

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.
1
Precursor of DNA synthesis: (deoxynucleoside triphosphate)

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

Note:

base + sugar = nucleoside

phosphate + base + sugar = nucleotide
2
UTP

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).

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).

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’

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).

Some ori are identified in bacteria (245
bp oriC in E. coli), yeast, chloroplasts
and mitochondria. ori is usually A-T
rich (easier melting).
3

DNA topoisomerase: unwinds the helix

DNA helicase: unzip the helix

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)
4
Synthesis of lagging strand. See Ref [2]

DNA polymerase requires an RNA primer to initiate DNA
synthesis, but RNA polymerase doesn’t need an RNA primer for
RNA synthesis.

In eucaryotes, repair system exists to ensure the fidelity of DNA
replication.

In E. coli, chromosome is circular, replication moves toward two
directions1.

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.
1
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).
5

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.

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.

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.

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)

5S, 5.8S, 18S, 28S in eucaryotes, extensively modified (e.g. methylation of the 2’OH position on ribose)


rRNA combines with some proteins to form ribosome
microRNA:

Catalytic RNA (ribozyme): enzymatically cleave RNA molecules.
6
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.

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’).

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.

When the termination sequence is reached, the enzyme stops adding nt to the RNA chain,
releases the product and dissociates from the DNA template.
7
Note:

RNA pol consists of the following subunits

2  subunits: enzyme assembly and promoter recognition

 and ’ subunits: catalytic center

 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.

Two strands of DNA

coding strand (sense strand): has the same sequence as the mRNA, often the sequence shown
in literature.


template strand (antisense strand):
Rifampicin, an antibiotic used against tuberculosis, inhibits transcription.
Differences in procaryotic and eucaryotic genes:
Procaryotic genes
8

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

LacY: encodes permease, transports -galactosides into the cell.

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.
9
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.

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 (<200 nt) complexed to
proteins called snRNPs (small nuclear ribonucleoprotein particles).
The splicing occurs at the junctions of
exon-intron; almost all introns begin with GU and end with AG.
10
Alternative Splicing

An primary transcript may undergo alternative splicing events, i.e. the mRNA may be
spliced one way in one tissue, but in different way in another tissue.

The exon skipping mechanism generates different gene products in different tissues from
the same structural gene. (also
facilitates the evolution of novel proteins
thanks to recombination).
Genetic Code [3]:

the blue print for any living cell,
universal, applicable to all
living system

3 bases on RNA codes for an
a.a

64 (43) combination: many of
which are redundant, e.g. UCU,
UCC, UCA and UCG specify
serine.

Nonsense codons: UAA, UAG,
UGA, don’t encode any thing
but acting as the stop signal in
translation.

Start codon: AUG (Met)
See Ref [4]
11
V. Translation [2]:

Requires the interaction of mRNA, charged tRNA, ribosomes and
some factors that facilitate the initiation, elongation and
termination.

tRNA: (also expressed by genes)

transports a.a. to the polypeptide chain

75~93 nucleotides

has a 2’ structure → folded
12

a particular a.a. is linked by its
carboxyl end (to form aminoacyl
group), after addition of the a.a, the
tRNA is “charged”.

anticodon determines what specific
a.a can be added.
1. Initiation: (usually the rate limiting step)
Procaryote

mRNA binds to the small (30S)
ribosomal subunit. The binding is
mediated by Shine-Delgarno (SD)
sequence (6-8 nt, -AGGAGGU-, RBS).
The SD is 5-10 nt upstream of the AUG
codon and is complementary to a
sequence of nt near the 3’ end of the 16S
rRNA of the small ribosomal subunit.

Several initiation factors (IF1, IF2 and
IF3) are required for initiation. E.g. IF2
is required for attachment of the first
aminoacyl-tRNA.

A formyl-methionine charged tRNA
binds to an mRNA-small ribosomal
complex. (Procaryotic cells possess 2
distinct methionyl-tRNA, one for
initiation (tRNAiMet), one for
incorporating Met into the interior of a
polypeptide (tRNAMet).

Large (50S) ribosomal subunit joins to
complete the initiation complex assembly.
13
Eucaryote

mRNA is transported out of nucleus for
translation.

Require at least 10 initiation factors
(eIF), several of which bind to the 40S
subunit.

The initiator tRNAiMet also binds to the
40S subunit prior to its interaction with
the mRNA, these and other eIFs form
the 43S complex (shown on the left) which is recruited to the mRNA with the help of eIFs that
are already bound to the mRNA.


The eIFs bound to the mRNA:

Poly A binding protein (PABP): binds the 3’ poly(A)

eIF4E: binds to the 5’ cap

eIF4A: a helicase, moves along the 5’ end unwinding the mRNA

eIF4G: serves as a linker between the 5’ capped end and the 3’ poly(A) end of the
mRNA, thus eIF4G converts a linear mRNA into a circular loop.
Once the 43S complex binds to the 5’ end, the complex scans along the transcript until it
reaches the first AUG. Once the 43S complex reaches the AUG codon, eIF2-GTP is
hydrolyzed to eIF2-GDP, and released along with other factors, and the large (60S)
subunit joins to complete the initiation.

Usually, but not always, the first AUG to be encountered is the initiation codon.
However, the AUG triplet is not sufficient to determine whether it is the start codon, it is
recognized efficiently as the initiation codon only when it is in the right context. An
initiation codon may be recognized in the sequence NNNPuNNAUGG (e.g. Kozak
sequence -CCACCAUGG-). The purine (A or G) at -3 position and the G immediately
following AUG can influence the translation efficiency by 10X.
14

This scanning mechanism is found in most eucaryotic cells, but some viruses (e.g.
poliovirus) use an alternative mechanism by which the 40S subunit associates directly
with IRES.

When the leader sequence is long, further 43S subunits can recognize the 5’ end before
the first has left the initiation site, creating a queue of subunits proceeding along the
leader to the initiation site.
2. Elongation: (similar for eucaryotes and procaryotes)
1.
The second codon (CUG) base pairs with the anticodon (GAC) of Leu-tRNA
2.
The Met of the initiator tRNA is joined to the Leu of Leu-tRNA by a peptide bond, and
the uncharged initiator tRNA is ejected from the ribosome.
3.
Translocation of the peptidyl-tRNA and the mRNA to the peptidyl site (P site, where
peptidyl-tRNA is, on the ribosome) from the aminoacyl site (A site), which opens the
aminoacyl site for the next codon (UUU).
4.
The third codon (UUU) base pairs with the anticodon (AAA) of Phe-tRNA
5.
The Leu of the peptidyl-tRNA is joined to Phe of the Phe-tRNA by a peptide bond, and
the uncharged Leu-tRNA is ejected from the ribosome.
6.
Translocation of the peptidyl-tRNA and mRNA to the peptidyl site from the aminoacyl
site, which opens the aminoacyl site for the next codon and codon-anticodon interaction.
15
3. Termination:

The above process continues until a stop codon
(UAA, UAG or UGA) is reached.

The stop codon binds with a termination factor,
the last tRNA is cleaved from the peptide chain
and ejected. The mRNA and the peptide are
released; the ribosomes are prepared for
recycling.

In many cases, Met is removed later.
Note:

Correct protein sequence is synthesized only if
the message is read in correct “reading frame”.
Every sequence can have 3 possible ORF
e.g. The big red dog
frameshift 

correct reading frame
HEB IGR EDD…
wrong, meaningless
Translation can occur co-transcriptionally in procaryotes, but in eucaryotes mRNA is
transported to ribosome for translation.
VI.
POST-TRANSLATIONAL PROCESSING

After translation, the polypeptide is further processed to be functional.

Many eucaryotic proteins require post-translational processing to be functional, while in
E. coli many of the post-translational processing steps are not performed.

Several types of processing:
1.
Folding into proper conformation, which can be assisted by chaperone proteins (e.g.
GroEL/GroES in E. coli)
2.
Signal peptide cleavage: many proteins carry signal sequences for intracellular
translocation and secretion, these signals are cleaved during secretion.
3.
Glycosylation: addition of sugar to the protein; very common in eucaryotic proteins;
could affect the protein function, stability, immunogenicity.
16
4.
Phosphorylation
5.
Proteolytic processing: e.g. cleavage of a polyprotein (VP) to form VP2 and VP4
(IBDV)
VII. Regulation of mRNA Transcription

Cells transcribe a common set of genes (housekeeping) that maintain routine cellular
functions, but not all genes are transcribed and translated at the same rate and the same
time. Genes are turned on and off when needed, otherwise cell resources would be
depleted. Therefore, gene expression is regulated. If a protein is required by a cell, a
signaling system initiates transcription of the pertinent genes.
In Bacteria

Gene clusters are controlled by a single promoter. Each gene has its own ATG codon.

In the promoter region, there are two binding sites for
The distance separating -35 and -10 sites is

The affinity of the promoter region to RNA polymerase is called the strength.

The sequence between TATA box and the initiation site is called
Example of Negative Regulation
17

Repressor protein binds to the operatorRNA pol cannot move along off (but there could
be a basal level of expression).

Ex: LacI represses Lac operon.
Effector (or called inducer) molecules bind to the repressor and release from the operator
region on (induction). e.g. the lacZ gene is off w/o -galactoside, when the substrate is
added, the enzyme activity appears within 2-3 min IPTG (isopropyl-beta-Dthiogalactopyranoside), a synthetic analogue of lactose, induces lac genes very efficiently.
Example of Positive Regulation: (increases the frequency of transcription)

Activator-operator complex attracts RNA pol and increases the transcription efficiency.

The same effector can be used in positive or negative control for different genes, i.e., it
may increase or decrease the transcription rates.
In Eucaryotes

Many genes are strictly regulated and may be transcribed in restricted types of cells, e.g.
 and  subunits of hemoglobin are only expressed in red blood cells. The
transcriptional regulation of genes is essential for maintaining cell specificity and
conserving cellular energy and function.
18

In general the gene expression in eucaryotic cells is controlled at the initiation of
transcription. Regulation at subsequent stages of transcription is relatively rare in eucaryotes.

The transcription is mediated by transcription factors (TF), many of which bind to DNA
sequences that are often <10 bp in length (boxes, elements, enhancers2).

Basal factors: Together with RNA
pol, bind at the start point and TATA
box.

Activators: TF that can bind the
promoters or enhancers and increase the
efficiency the basal apparatus binds to the
promoter. Some activators are ubiquitous
but others have a regulatory role and are
synthesized/activated at specific time or
in specific tissues, to bind the response
elements.

Coactivators: do not bind the DNA
themselves, provide a connection
between activators and the basal
apparatus.

Some regulators act to change the
chromatin structure. (see Appendix)

p53 is a transcription
factorupregulate p21 and p16 cell
cycle regulation mutation of p53 often
leads to cancer.

Each eucaryotic gene has its own set of elements within the promoter, but there are
common elements within a typical promoter (200 bp): Active promoters contain at least one of these
three, but may not contain all three

TATA box (-25 bp): 8 bp
containing a TATA sequence, binds general transcription factors (TFIID for pol II
transcription), responsible for correctly locating the transcription start site.

2
CCAAT box (cat box, -75): binds TF CTF and NF1, affects the efficiency
Enhancers: elements that can stimulate the transcription. They may be located a considerable distance from the
transcription start site, and may be upstream or downstream of the gene they control. Folding and bending of DNA might
bring these regions close to each other.
19


GC box (-90 bp): repeated GC nt (GGGCGG). Binds TF SP1.
Some genes contain the response elements (a common promoter or enhancer element that
can respond to a certain factor). e.g. HSE (heat shock element).
Initiation Process

TF IID binds to TATA box, other transcription factors
subsequently bind to form a protein complex.

RNA pol II then binds (oriented toward the gene) the
complex. With the aid of additional TF, the transcription
is initiated at the correct starting point.
Note:

Transcription control is generally positive in eucaryotes
. Repression is usually
accomplished at the level of unavailability or inactivation
of TF, change in
In mammals, the methylation of DNA occurs at the cytosine bases in CpG dinucleotide via the
methyltransferase [1]. A high CpG content is found in regions known as CpG islands (a
stretch of DNA 1-2 kb that has clusters of CpG doublets). CpG islands surround the
promoters of constitutively expressed genes where they are unmethylated. Methylation of a
CpG island prevents activation of a promoter within it. The methylation can recruit HDAC
activities to methylated CpG sequences to establish a repressed state of chromatin.
(epigenetics) .
20
VIII. Summary
Eucaryotic promoter elements and other sequences important for transcription and
translation [3].

Which strand serves as the template depends on the promoter and direction the RNA pol
is moving.
RNA pol
5’
pol
3’

3’
5’
RNA synthesis must be 5’ to 3’ the DNA template must be traversed from 3’ to 5’
So

If RNA pol moves to right top is sense, bottom is template (antisense)

If RNA pol moves to leftbottom is sense, top is template

On one end of chromosome, the top strand may be template, on the other locus
the bottom may be the template.
21
Appendix

Chromatin: complex of histones, DNA and other proteins

The nucleus of an interphase (G1, G2, S phases) cells contains the chromatin fiber whose
extended state is more suitable for gene transcription, only when the cells enter M phase
do the chromatin fibers compact to form the chromosome.
22

The metaphase chromosome has undergone DNA replication, so both chromosomes are
sister chromatids (same chromosomes after replication). After replication, they are
aligned in parallel and then linked by centromere.
Alternative Splicing [4]
23
References:
[1]
Krauss G. Biochemistry of signal transduction and regulation. Weinheim: Wiley-VCH
Verlag, 2003.
[2]
Lewin B. Genes VIII. Upper Saddle River, NJ. : Pearson Prentice Hall, 2004.
[3]
Barnum SR. Biotechnology: an Introduction. New York: Thomson Learning, 2005.
[4]
Watson J, Myers RM, Caudy AA, Witkowski JA. Recombinant DNA: Genes and
Genomes. New York: W.H. Freeman and Company, 2007.
[5]
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the
Cell. New York: Garland Science, 2002.
24