Nucleic acid (macro-molecules):

Determining the correct order amino

acids sequence structure and
function

One ribosomal RNA transcription

Lodish Berk Kaiser Krieger Scott Bretscher Ploegh Matsudaira

MOLECULAR CELL
BIOLOGY
SIXTH EDITION

CHAPTER 4
Basic Molecular Genetic
Mechanisms
Copyright
2008 W.
H.Freeman
andand
Company
2008
W. H. Freeman
Company

Gene (DNA) contains all information

build the cells and tissues of
organism
Deoxyribonucleic acid (DNA) contains the
information prescribing the amino acid
sequence of proteins
This information is arranged in units termed
genes
Ribonucleic acid (RNA) serves in the cellular
machinery that chooses and links amino acids
in the correct sequence
The central dogma: DNA RNA Protein
DNA and RNA are polymers of nucleotide
subunits

RNA + ribose + protein

ribosomal ribonucleoprotein
complexes (rRNPs)

Chemical structure of the principal bases (ch3)

Monomerspolymers

DNA: ATCG
RNA: AUCG

Four basic molecular genetic processes:

Protein synthesis: 1 to 3
rRNA: ribosomal RNA;
tRNA: transfer RNA
rNTPS: ribonucleoside triphosphate monomers;
dNTP:deoxyribonucleoside triphophate

Structure of nucleic acid

A nucleic acid strand is a linear polymer with end to end directionality
REMEMBER:
DNA = deoxyribonucleotides;
RNA = ribonucleotides (OH-groups at
the 2 position)
Note the directionality of DNA (5-3 & 35) or RNA (5-3)
DNA = A, G, C, T ; RNA = A, G, C, U

Nucleotide subunits are linked together by phosphodiester bonds

Native DNA is a double helix of complementary antiparallel

strands
1953, Watson and Francis: proposed
that DNA has a double-helical
structure Nature, 4356, 737-728 (1953)
DNA consists of two associated
polynucleotide strands that wind
together to form a double helix.
53; 35 antiparallel
Base pair: H-bond formation, A-T (2)
and G-C (3)
Complementary: two polynucleotide
consequence of the size, shape and
chemical composition, by base pair
interaction (A-T and C-G)
There are two major forces that
contribute to stability of helix formation:
Hydrogen bonding in base-pairing
Hydrophobic interactions in base
stacking ()

Nucleic acid as hetero-polymers

Nucleosides, nucleotides

(Ribose sugar, (2-deoxy ribose sugar,

RNA precursor)
DNA precursor)

(2-deoxy thymidine triphosphate, nucleotide)

DNA and RNA strands

So
DNA

RNA

Most DNA in cells is a right handed

helix
X-ray data of DNA: (B-form)
1.The stacked bases are regularly
spaced 0.34-0.36nm
2.Helix makes a complete turn every
3.6nm, about 10.5 pairs per turn.

B-DNA

A-DNA

B DNA most common d(CGCGAATTCGCG)d(CGCGAATTCGCG)

A DNA, in low humidity condition, B transform to A form; RNA-RNA,
RNA-DNA d(AGCTTGCCTTGAG)d(CTCAAGGCAAGCT)
Z DNA, short DNA molecules composed of alternating purine-pyrimidine
nucleotides (GC), right transform to left
d(CGCGCGCGCGCG)d(CGCGCGCGCGCG)

Z-DNA

DNA compositional biases

B-DNA

A-DNA

R.H. helix

R.H. helix

Z-DNA

Base compositions of genomes: G+C (and therefore also

A+T) content varies between different genomes
The GC-content is sometimes used to classify organism in
taxonomy
High G+C content bacteria: Actinobacteria
e.g. in Streptomyces coelicolor it is 72%

Low G+C content: Plasmodium falciparum (~20%)

Other examples:
Saccharomyces cerevisiae (yeast)

38%

Arabidopsis thaliana (plant)

36%

Escherichia coli (bacteria)

50%

L.H. helix

TBP protein can binds to

the minor groove of specific
DNA (rich AT)
untwisting and sharply
bending the double helix
transcription ability
Why is rich AT region ?

Why RNA degradation more easy than DNA???

DNA

RNA

Base-catalyzed hydrolysis of RNA

2-OH site as a nucleophile at normal

pH attacking phophodiester bond
degradation
In 2 site, DNA is more stable than RNA.

DNA can undergo reversible strand separation (denaturation)

Tm: melting temperature

G-C more need more energy
Denature of single stranded DNA random coil (without organized structure)
Renature vs hybribization

SV40 viral DNA

Many prokaryotic genomic DNA
and viral DNA are circular
molecules.
Circular DNA molecules in
eukaryotic mitochondria and
chloroplasts
Circular DNA without end, when
replication: open DNA
unwinding DNA torsional (
) stress winding ()
formed super-coil ()
Topoisomerase I (bacterial and
eukaryotic cell has) bind to
DNA
breaks a phosphodiester bond in one strands DNA formed nick loss
supercoiled ligates the two ends of the broken strand.
Topoisomerase II, breaks two strands DNA

Supercoils
Supercoiling of DNA can only occur in
closed-circular DNA or linear DNA where
the ends are fixed.

Underwinding produces negative supercoils,

whereas overwinding produces positive supercoils.

Supercoiling induced by separating the strands

of duplex DNA (eg., during DNA replication)

DNA (double strain) open single strain replication or transcription

spuercoiling need topoisomerase

Transcription of protein-coding genes and formation of functional mRNA

Relaxed and supercoiled plasmid DNAs

DNA RNA Protein function
ATCG

AUCG
mRNA
tRNA
rRNA

Encode:

AA
protein coding gene

gene mRNA protein

DNA replication Direction 5 to 3 ~800 nd/sec

RNA polymeration: Direction 5 to 3 ~40 nd/sec
Translation Direction 5 to 3 ~15 aa/sec

Different types of RNA exhibit various conformations related

their functions
AUCG: CG has 3 H-bond
Most RNA are single strand
Various RNA carry out specific
functions
Eukaryotic cell, RNA self-splicing
> Secondary structure H-bond
dependent

5-10 nucleotides

>10 nucleotides

Three Different Classes of RNA

1) rRNA (ribosomal)
large (long) RNA molecules
structural and functional components of ribosomes
highly abundant
2) mRNA (messenger)
typically small (short)
encode proteins
multiple types, not abundant
3) tRNA (transfer) and small ribosomal RNAs
very small
Important in translation
Not all genes encode proteins

DNA
DNA
Deoxyribonucleic acid
ATCG
More rigid
More stable

RNA
Ribonucleic acid
AUCG
More flexible
More unstable
mRNA, rRNA, tRNA

transcription

RNA

A template DNA strand is transcribed into a

complementary RNA chain by RNA.
Ribonucleoside triphosphate (rNTP) are polymerized to
form a complementary RNA by RNA polymerase.
Polymerization involves a nucleophilic attack by the 3
oxygen in the growing RNA chain on the a
phosphate of the next nucleotide formed
phosphodiester bond and release pyrophosphate
Direction: 5 3; opposite in polarity to their template
DNA strands
DNA AU TA C G GC transcribed to
RNA

Release PPi

The micro RNA (miRNA):

1. Regulate specific mRNA
2. Produced by RNA polymerase

RNA polymerase begins transcription is +1

Downstream: +,
Upstream: -

pri-miRNA
Drosha RNaseIII
pre-miRNADroshapri-miRNA
Exportin-5 pre-miRNA

miRNA duplex pre-miRNA

(20~22 )
mature miRNAmiRNA

Bacterial (Prokaryotic) Transcription

Three stages in transcription

Promoters
- DNA sequences that guide RNA polymerase to the beginning of
a gene (transcription initiation site).
Terminators
- DNA sequences that specify then termination of RNA synthesis
and release of RNAP from the DNA.

Need transcription factor help

Many transcription factor

binding for help RNA
polymerase binding

RNA Polymerase (RNAP)

- Enzyme for synthesis of RNA.
Reaction (ordered series of steps)
1) Initiation.
2) Elongation.
3) Termination.

About 14 base pairs

Recognition

rNTP vs.dNTP

Three stages in transcription

Termination of transcription
Two mechanisms
1) Rho - the termination factor protein

About 8 base pair

For continuous RNA synthesis and
without dissociation

rho is an ATP-dependent helicase

it moves along the RNA transcript, finds the "bubble",
unwinds it and releases the RNA chain.

2) Rho-Independent
- termination sites in DNA
inverted repeat, rich in G:C, which forms a
stem-loop in RNA transcript

Rho-Dependent Transcription Termination

(depends on a protein AND a DNA sequence)

G/C -rich site

Termination of transcription

Two mechanisms
2) Rho-Independent
- termination sites in DNA

RNAP slows down

Rho helicase
catches up

Elongating complex is disrupted

inverted repeat, rich in G:C, which forms a

stem-loop in RNA transcript

Rho-independent
transcription
termination

Rho-Independent Transcription Termination

(depends on DNA sequence - NOT a protein factor)

RNAP pauses when it reaches

a termination site.
The pause may give the
hairpin structure time to fold
The fold disrupts important
interactions between the RNAP
and its RNA product
The U-rich RNA can dissociate
from the template

Stem-loop structure

DNA
A
T
G
C

Transcriptional mechanism-1

Bacterial RNA polymerase

RNA
T The complex is now disrupted
U and elongation is terminated
C
G

RNA Polymerase

Structure of RNA polymerase

RNA polymerase are similar in eukaryotic

and prokaryotic cell
Five subunit:
2 large subunit: , ; 2 smaller subunits
and (only Stabilizes and assembly
of its subunits)
Only a single RNA polymerase
(prokaryotic)
In E.coli, RNA polymerase is 465 kD
complex, with 2 , 1 , 1 ', 1
' binds DNA
2+
binds rNTPs and interacts withMg

and ' together make up the

active site
subunits appear to be essential for
assembly and for activation of
Current model of bacterial RNA
enzyme by regulatory proteins
polymerase bond to a promoter

Different Types of RNA Polymerase

In Bacteria (simple system)
- all three classes are transcribed by the same RNA polymerase
(RNAP for short)
In Eukaryotes (complex system)
- each class is transcribed by a different RNA Polymerase
RNAP I - rRNAs
RNAP II - mRNAs
RNAP III - tRNAs & small ribosomal RNAs

Remember: only RNAP did not transcript !!!! Need many

transcription factor (protein)
FlashFlash-2

1
2
3
4
5

RNA Polymerase is a spectacular () enzyme, functioning in:

Recognition of the promoter region
Melting of DNA (Helicase + Topisomerase); unwinding DNA
RNA Priming (Primase)
RNA Polymerization; add rNTP
Recognition of terminator sequence

RNA-DNA hybrid Length? 3 to 9 bases, it is short and transit

In Bacterial which can hold~16 bp

In yeast which can hold ~25 bp

Thus, RNAP is a multisubunit enzyme

One model for transcriptional activation

Gene Regulation
Protein complex DNA open/tight DNA transcription
Transcription is regulated by proteins binding to or near
promoters
Three types of proteins involved:
Specificity factors
Repressors
Activators
Repressors: bind to specific sites on DNA
Called operators
Either near or overlapping the promoter
Block movement of RNA-polymerase
-Activators: bind to specific sites on DNA, help RNAP moving
Operon: arrangement of genes in a functional group

Organization of genes differs in prokaryotic and eukaryotic DNA

Genomes

In prokaryotic:
1. logic: genes devoted () to a
single metabolic goal; protein synthesis
from a contiguous array in DNA. It
means that one gene one protien.
one
operon one goal (function)
2. Arrangement of genes in a functional
group is cell an operon, because it
operate as a unit from a single
promoter. One promoter one gene (
or genes) one protein (or proteins)
3. The genes are closely packed with very
few non-coding gaps
DNA direct to co-linear mRNA
translated protein

Eukaryotic precursor mRNA are processed to form functional mRNAs

In eukaryotic
1. RNA discontinuous in
corresponding DNA sequence
2. DNA contain exons (coding
sequence) and introns (nonprotein-coding segments)
3. DNA RNA, remove introns and
carefully stitched back together to
produced many mRNAs
4. Functional (mature) mRNA from
precursor mRNA processed
(splicing)
5. DNA pre-mRNA splicing
mature mRNA protein add
modification mature protein
Each gene is transcripbed from its own promoter
Tryptophan (trp)

Tryptophan metabolite enzyme

In prokaryotic, protein synthesis can occur in 5 or 3

end of mRNA; transcription and translation can
occur at the same time.
In eukaryotic, in nucleus DNA transcription
precursor mRNA procession functional
mRNA transport to cytoplasm translated to
protein; Transcription and translation are in
different time and place.
Pre-mRNA are modified at the tow ends, and keep in
mRNA. It can protect the degradation of RNA form
nucleus to cytoplasm. Dont need DNA template.
Modification of 5 end: by RNA polymerase II add
5cap; methylation
Modification of 3 end: by poly A polymerase, add
100-250 A and produced poly A tail.
mRNA processing RNA splicing, 5 and 3 retain
noncoding regions (untranslated regions; UTRs).
In mammalian mRNA, 5 UTR about >100
nucleotides, 3 UTR about several kilobases
The ribose of the second nucleotide also is methylated

Alternative RNA splicing increase the number of proteins expressed

from a single eukaryotic gene

RNA Processing:
Prokaryotes: transcription and translation can be

One gene RNA splicing

different RNA different protein
Isoform: by alternative splicing
production of different forms of a
protein.
Untranslated region
One gene can lead to more than one
protein (e.q. antibodies)

concurrent.
Eukaryotes: Nucleus (RNA synthesis) and cytoplasm
(Protein synthesis) are separated.
Primary transcript undergoes several modifications.
5 cap is added to 5 nucleotide; m7Gppp (Stability)

Exons: part of the gene that is expressed.

Introns: part of gene that is spliced out
from pre-mRNA.

String of adenylic acids are added to the 3 end (Poly

A tail)

Sometimes some exons are also spliced

out.

Splicing: internal cleavage to excise introns followed

by ligation of coding exons
Formed three protein-coding exon

5 and 3 ends of eukaryotic mRNA

Functions of 5 cap and 3 polyA

Both cap and polyA contribute to stability of mRNA:
Most mRNAs without a cap or polyA are degraded
rapidly.
Shortening of the polyA tail and decapping are part of
one pathway for RNA degradation in yeast.
Need 5 cap for efficient translation:

Add a GMP.
Methylate it and
1st few nucleotides

Cut the pre-mRNA

and add As

Eukaryotic translation initiation factor 4 (eIF4)

recognizes and binds to the cap as part of initiation.
Assists mRNA export to the cytoplasm

ch6 p216

p249

Gene: DNA regions encoding proteins or functional RNA

Intron: non-functional DNA, non-coding regions of DNA
Extron: functional DNA, coding region of DNA
Transposable (mobile) DNA: non-coding region, repeat, evolutionary
DNA must be contend: human cell has 2 meters DNA!!!!!SO must be
highly compacted
In eukaryotes, DNA + protein chromatin chromosome
histone

The structure of genes and chromosomes

Eukaryotic gene structure

A gene: as the entire nucleic acid sequence that is necessary for the
synthesis of a functional gene product
Coding region: coding amino acids sequence, or functional RNA
Enhancer: transcript regions, not coding region; it regulated
transcriptive activity
Most eukaryotic genes contain introns and produce mRNA
encoding single proteins
Simple and complex transcriptions units are found in eukaryotic cells
Cistron: a genetic unit encoding a single polypeptide
Polycistron: a genetic unit (not a only a gene) encoding multiple
polypeptides; also called operon, like prokaryotic cell for live
Most eukaryotic cell has mono-cistron.
Prokaryotes have compact genomes and their transcripts often contain
multiple protein coding regions (called open reading frames or ORFs)
These mRNAs are called polycistronic mRNAs (a cistron is a concept
that is similar to a gene, and for many genes the cistron=gene)

Homologous recombination: meiosis

Exon 3 is lost

L: non-coding repeat, also called Transposable (mobile) DNA

its easy to homologous recombination

Homologous recombination and generate genetic diversity

Generate genetic diversity

among the individuals of
a species by causing the
exchange of large regions
of chromosomes between
the maternal and paternal
pair of homologous
chromosomes during the
cellular division the
generates germ cells

Simple and complex eukaryotic transcription

Mutation control region: no mRNA expression no protein no function

Mutation Exon : mRNA expression (some wrong) abnormal protein activity change

For gene that are transcribed from

different promoters (regulator
factor) in different cell type

Protein-coding genes may be solitary or belong to a gene family

Solitary gene: in multicellular organism, 20-50% protein coding gene are reprsented
Duplicated gene: gene family protein family homologous
duplicated gene encode protein with similar

New Roles of RNA

RNAi - RNA interference
siRNA- active molecules in RNA interference; degrades
mRNA (act where they originate)
miRNAs - tiny 2124-nucleotide RNAs; probably acting as
translational regulators of protein-coding mRNAs
stRNA - Small temporal RNA; (ex. lin-4 and let-7 in
Caenorhabditis elegans
snRNA - Small nuclear RNA; includes spliceosomal
RNAs (processing)
snoRNA - Small nucleolar RNA; most known snoRNAs
are involved in rRNA modification

Alternative RNA splicing increases the number or proteins expressed

from a single eukaryotic gene

Production of heavy chain genes in mouse by recombination of V, D,

J, and
C gene segments during development

Higher eukaryote have multidomain tertiary

structure only from a small number of exons.
Single gene Multiple intronsalternative
splicing protein isoforms
Alternative splicing: The presence of multiple
introns in many eukaryotic genes permits
expression of multiple, related proteins form a
single gene.
> 20 isoforms fibronectin from different
alternatively spliced mRNA

Cell type specific splicing of fibronectin pre-mRNA

Alternative splicing
Different mRNAs
can be produced by
same transcript

Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

Differences Between Transcription In Prokaryotes and

Eukaryotes
Transcription And Translation In Prokaryote-----------the same time
Eukaryotic Transcription and translation--------------different time
Processing Eukaryotic mRNA