Download •MOLECULAR CELL BIOLOGY

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)
Monomers→polymers
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
專有名詞要不止要
放在心中,更要放
在腦中
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’ & 3’5’) 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.
5’→3’; 3’→5’ 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 (堆)
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%
鏈黴菌
Low G+C content: Plasmodium falciparum (~20%)
瘧原虫
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 (扭
力) stress → winding (纏繞) →
formed super-coil (超螺旋)
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 A→U T→A C→ G G→C 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 ：由基因組中轉錄出來
Drosha ：一種RNaseIII
pre-miRNA：由Drosha切割pri-miRNA 而來
Exportin-5 ：可將pre-miRNA由細胞核運到
細胞質。
miRNA duplex ： pre-miRNA被切割後的產物
(20~22個鹼基 )
mature miRNA：有活性的單鏈miRNA
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
初期
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: β, β’; 2 smaller subunits
α and ω (only Stabilizes and assembly
of its subunits)
Only a single RNA polymerase
(prokaryotic)
In E.coli, RNA polymerase is 465 kD
complex, with 2 α, 1 β, 1 β', 1 σ
β' binds DNA
2+
β binds rNTPs and interacts withMg
σ
β and β ' together make up the
active site
α 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 (壯觀) 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 (致力於) to a
single metabolic goal; protein synthesis
from a contiguous array in DNA. It
means that one gene → one protien→.
可以多段有功能基因連在一起 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. Don’t need DNA template.
Modification of 5’ end: by RNA polymerase II → add
5’cap; 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 A’s
– Eukaryotic translation initiation factor 4 (eIF4)
recognizes and binds to the cap as part of initiation.
– Assists mRNA export to the cytoplasm
轉到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 21–24-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 introns→alternative
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