Download Fundamentals of Nucleic Acid Biochemistry: RNA

Fundamentals of Nucleic
Acid Biochemistry: RNA
Donna Sullivan, PhD
Division of Infectious Diseases
University of Mississippi Medical Center
DNA AND RNA: BASIC
STRUCTURE
Repeating Nucleotide Subunits In DNA
and RNA
STRUCTURE OF RNA

SUGAR



Ribose
Phosphate group
Nitrogen containing base
Adenine
 Guanine
 Cytosine
 Uracil

THE NUCLEOTIDE: RNA
OH
O=P-O-5CH2
BASE
OH
O
4C
H
1C
H
3C
OH
H
2C
0H
H
Adenine
Guanine
Cytosine
Uracil
The Forms Of RNA: Not Just
Another Helix


Does not normally exist as a double helix,
although it can under some conditions
Can have secondary structure:
Hairpins: pairing of bases within 5-10 nt
 Stem-loops: pairing of bases separated by >50 nt


Can have tertiary structure:

Pseudoknot, cloverleaf
RNA Structures
Structure Of RNA
Structure of tRNA
Structure of rRNA
RNA Replication vs. DNA
Replication




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RNA replication:
Requires no priming
Has many more initiation sites
Is slower (50–100 b/sec vs. 1000 b/sec)
Has lower fidelity
Is more processive
Transcription




Transcription is the enzyme-dependent process
of generating RNA from DNA.
The process is catalyzed by a DNA-dependent
RNA polymerase enzyme.
Only “coding” segments of DNA (genes) are
transcribed.
Types of genes include structural genes (encode
protein), transfer RNA (tRNA), and ribosomal
RNA (rRNA).
Transcription


Transition from DNA to RNA
Initiation: Gene recognition






RNA polymerase enzyme and DNA form a stable complex at the gene
promoter.
Promoter: Specific DNA sequence that acts as a transcription start site.
Synthesis of RNA proceeds using DNA as a template.
Only one strand (coding strand) is transcribed, the other strand has
structural function.
Transcription factors are proteins that function in combination to
recognize and regulate transcription of different genes.
Termination signal
RNA POLYMERASES

Cellular RNA polymerase
RNA pol I transcribes rRNA genes
 RNA pol II transcribes protein encoding genes
 RNA pol III transcribes tRNA genes


Viral RNA polymerases
Reverse transcriptase of retroviruses
 RNA dependent RNA polymerases of negative
stranded viruses

RNA Polymerase Enzymes
(DNA-dependent RNA Polymerase)




RNA Polymerase I transcribes most rRNA
genes (RNA component of ribosomes).
RNA Polymerase II transcribes structural genes
that encode protein.
RNA Polymerase III transcribes tRNA genes
(for transfer RNAs).
RNA Polymerase IV is the mitochondrial RNA
polymerase enzyme.
RNA TRANSCRIPTION
General Organization of a Eukaryotic Gene
Promoter/Enhancer
Enhancer Region
Regulatory
Proteins
Promoter Region
Regulatory
Proteins
RNA
TFIID Polymerase II
TATA
200–2000 bp
100–200 bp
200–10,000 bp
~30 bp
Gene Structure
Upstream
5´
Downstream
Exon
1
Promoter
Intron
1
5´- UTR
(Untranslated
Region)
Exon
2
Intron
2
Exon
3
Intron
3
Exon
4
3´
3´- UTR
(Untranslated Region)
Polyadenylation
Signal
Nuclear Processing of RNA



Chemical modification reactions (addition of
the 5´ CAP)
Splicing reactions (removal of intronic
sequences)
Polyadenylation (addition of the 3´ polyA tail)
Processing of mRNA


In eukaryotes, the mRNA molecule that is
released after transcription is called precursor
mRNA or pre-mRNA.
It undergoes several changes before being
exported out of the nucleus as mRNA.
Processing of mRNA


5’ end is capped with a modified form of the G
nucleotide known as the 5’ cap
At the 3’ end, an enzyme adds a long series of A
nucleotides referred to as a poly-A tail
It serves to protect the mRNA from enzymes in the
cytoplasm that may break it down
 The greater the length of the poly-a tail, the more
stable the mRNA molecule

RNA Transcription and
Processing


The process of RNA transcription results in the
generation of a primary RNA transcript
(hnRNA) that contains both exons (coding
segments) and introns (noncoding segments).
The noncoding sequences must be removed
from the primary RNA transcript during RNA
processing to generate a mature mRNA
transcript that can be properly translated into a
protein product.
RNA Processing



Capping of the 5´-end of mRNA is required for
efficient translation of the transcript (special
nucleotide structure).
Polyadenylation at the 3´-end of mRNA is
thought to contribute to mRNA stability (PolyA
tail).
Once processed, the mature mRNA exits the
nucleus and enters the cytoplasm where
translation takes place.
RNA Processing
Biogenesis of Mature mRNA
Genomic
DNA
Boundary
Transcriptional
Promoter
Exon 1
Intron 1
Exon 2
Intron 2 Exon 3
Gene Structure in Chromosomal DNA
Transcription
Initiator
ATG
Stop PolyA
Codon Signal
Primary RNA
Transcript
Posttranscriptional
Processing
Initiator
ATG
Mature mRNA
Transcript
Stop
Codon
PolyA Tail
Genomic
DNA
Boundary
RNA Processing
Fundamentals of RNA Splicing
Primary RNA Transcript
Exon 1
Intron 1
Exon 2
…NNAG GUAAGU
CAG GNNN…
Exon 1
Exon 2
5´ Splice Junction
3´ Splice Junction
RNA Splicing
Exon 1
Exon 2
Mature RNA Transcript
RNA Processing: Spliceosomes

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Ribonucleoproteins (snRNPs) function in RNA
processing to remove intronic sequences from the
primary RNA transcript (intron splicing).
Alternative splicing allows for the generation of
different mRNAs from the same primary RNA
transcript by cutting and joining the RNA strand at
different locations.
snRNPs are composed of small RNA molecules and
several protein molecules.
Five subunits form the functional spliceosome.
RNA Processing
Alternative Splicing of RNA
Normal
Splice
Site
Exon 1
Alternative
Splice
Site
Exon 2
RNA Splicing
Normal Transcript
Alternative Transcript
Exon 3
Structural Genes Encode Proteins


The majority of structural genes in the human
genome are much larger than necessary to
encode their protein product.
Structural genes are composed of coding and
noncoding segments of DNA.
Structural Genes Encode Proteins


The structure of a typical human gene includes
informational sequences (coding segments
termed exons) interrupted by noncoding
segments of DNA (termed introns).
The exon-intron–containing regions of genes
are flanked by non transcribed segments of
DNA that contribute to gene regulation.
Factors That Control Gene
Expression Include:
 Cis
elements
are DNA
sequences.
 Trans
elements
are proteins.
Control of Gene Expression



Primary level of control is regulation of gene
transcription activity.
TATA box contained within the gene promoter
provides binding sites for RNA polymerase.
Enhancer sequences can be sited very far away
from the gene promoter and provide for tissuespecific patterns of gene expression.
Gene Enhancer Sequences
5´
Exon
1
Intron
1
Enhancer
Promoter
5´-UTR
1. Enhancer sequences are usually sited a long
distance from the transcriptional start site.
2. Enhancers maintain a tissue-specific or cellspecific level of gene expression.
3. The gene promoter contains TATA box
upstream of transcription start site.
Protein binding sequences in the
‘Promoter’ region
Gene Regulation: Two types of
regulation

Negative regulation
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Substrate induction (lac operon): gene OFF unless substrate
is present
End product repression (trp operon): gene OFF if end
product is present
Common in bacteria
Positive regulation

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Gene is OFF until a protein turns it ON
Regulatory proteins turn gene ON
Occurs in eukaryotes
Negative Regulation
Positive Regulation
Lac Operon

Operon
Gene organization in bacteria in which several
proteins are coded by one mRNA
 Allows all proteins to be controlled together

Differences Between Prokaryotes
And Eukaryotes

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Prokaryote gene expression typically is regulated by an
operon, the collection of controlling sites adjacent to
protein-coding sequence.
Eukaryotic genes also are regulated in units of proteincoding sequences and adjacent controlling sites, but
operons are not known to occur.
Eukaryotic gene regulation is more complex because
eukaryotes possess a nucleus (transcription and
translation are not coupled).
Two “Categories” Of Eukaryotic
Gene Regulation


Short-term - genes are quickly turned on or off
in response to the environment and demands of
the cell.
Long-term - genes for development and
differentiation.
Eukaryote Gene Expression Is
Regulated At Six Levels
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Transcription
RNA processing
mRNA transport
mRNA translation
mRNA degradation
Protein degradation
Transcription Control Of Gene
Regulation Controlled By Promoters
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Occur upstream of the transcription start site.
Some determine where transcription begins (e.g.,
TATA), whereas others determine if transcription
begins.
Promoters are activated by highly specialized
transcription factor (TF) proteins (specific TFs bind
specific promoters).
One or many promoters (each with specific TF
proteins) may occur for any given gene.
Promoters may be positively or negatively regulated.
Transcription Control Of Gene
Regulation Controlled By Enhancers

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Occur upstream or downstream of
the transcription start site.
Regulatory proteins bind specific
enhancer sequences; binding is
determined by the DNA sequence.
Loops may form in DNA bound
to TFs and make contact with
enhancer elements.
Interactions of regulatory proteins
determine if transcription is
activated or repressed (positively or
negatively regulated).
Chromosome Structure, Eukaryote
Chromosomes, And Histones

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Prokaryotes lack histones and other structural proteins,
so access to the DNA is straightforward.
Eukaryotes possess histones, and histones repress
transcription because they interfere with proteins that
bind to DNA.
Verified by DNase I sensitivity experiments:


DNase I readily degrades transcriptionally active DNA.
Histones shield non-transcribed DNA from DNase I, and
DNA does not degrade as readily.
Chromosome Structure, Eukaryote
Chromosomes, And Histones


If you experimentally add histones and promoter
binding proteins; histones competitively bind to
promoters and inhibit transcription.
Solution: transcriptionally active genes possess looser
chromosome structures than inactive genes.
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Histones are acetylated and phosphorylated, altering their
ability to bind to DNA.
Enhancer binding proteins competitively block histones if
they are added experimentally with histones and promoterbinding TFs.
RNA polymerase and TFs “step-around” the
histones/nucloesome and transcription occurs.
Genomic Imprinting
(Silencing)
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Methylation of DNA inhibits transcription of some genes.
Methylation usually occurs on cytosines or adenines.
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5-methyl cytosine
N-6 methyl adenine
N-4 methyl cytosine
CpG islands are sites of methylation in human DNA.
CpG Island
…ggaggagcgcgcggcggcggccagagaaaaa
gccgcagcggcgcgcgcgcacccggacagccgg
cggaggcggg...
DNA Methylation And Transcription
Control

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Small percentages of newly
synthesized DNAs (~3% in
mammals) are chemically
modified by methylation.
Methylation occurs most often
in symmetrical CG sequences.
Transcriptionally active genes
possess significantly lower levels
of methylated DNA than
inactive genes.


A gene for methylation is
essential for development in
mice (turning off a gene also
can be important).
Methylation results in a human
disease called fragile X
syndrome; FMR-1 gene is
silenced by methylation.
Hormone Regulation: Example Of
Short-term Regulation Of Transcription
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Cells of higher eukaryotes are specialized
and generally shielded from rapid changes
in the external environment.
Hormone signals are one mechanism for
regulating transcription in response to
demands of the environment.
Hormones act as inducers produced by one
cell and cause a physiological response in
another cell.
Hormone Regulation: Example Of
Short-term Regulation Of Transcription
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Hormones act only on target cells with hormone
specific receptors, and levels of hormones are
maintained by feedback pathways.
Hormones deliver signals in two different ways:

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Steroid hormones pass through the cell membrane and bind
cytoplasmic receptors, which together bind directly to DNA
and regulate gene expression.
Polypeptide hormones bind at the cell surface and activate
transmembrane enzymes to produce second messengers
(such as cAMP) that activate gene transcription.
Examples Of Mammalian Steroid
Hormones
Hormone Regulation

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Genes regulated by steroid hormones possess binding
regions in the sequence called steroid hormone
response elements (HREs).
HREs often occur in multiple copies in enhancer
sequence regions.

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When steroid is absent: Receptor is bound and “guarded” by
chaperone proteins; transcription does not occur.
When steroid is present: Steroid displaces the chaperone
protein, binds the receptor, and binds the HRE sequence;
transcription begins.
Model Of Glucocorticoid Steroid
Hormone Regulation
RNA Processing Control
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RNA processing regulates mRNA production from
precursor RNAs.
Two independent regulatory mechanisms occur:
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Alternative polyadenylation = where the polyA tail is added
Alternative splicing = which exons are spliced
Alternative polyadenylation and splicing can occur
together.
Examples:
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Human calcitonin (CALC) gene in thyroid and neuronal cells
Sex determination in Drosophila
Alternative Polyadenylation And Splicing Of The
Human CACL Gene In Thyroid And Neuronal
Cells
mRNA Transport Control
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Eukaryote mRNA transport is regulated.
Some experiments show ~1/2 of primary
transcripts never leave the nucleus and are
degraded.
Mature mRNAs exit through the nuclear pores.
mRNA Transport Control
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Unfertilized eggs are an example, in which mRNAs (stored in
the egg/no new mRNA synthesis) show increased translation
after fertilization).
Stored mRNAs are protected by proteins that inhibit translation.
Poly(A) tails promote translation.
Stored mRNAs usually have short poly(A) tails (15-90 As vs 100300 As).
Specific mRNAs are marked for deadenylation (“tail-chopping”)
prior to storage by AU-rich sequences in 3’-UTR.
Activation occurs when an enzyme recognizes AU-rich element
and adds ~150 As to create a full length poly(A) tail.
mRNA Degradation Control
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All RNAs in the cytoplasm are subject to degradation.
tRNAs and rRNAs usually are very stable; mRNAs vary
considerably (minutes to months).
Stability may change in response to regulatory signals and is
thought to be a major regulatory control point.
Various sequences and processes affect mRNA half-life:
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AU-rich elements
Secondary structure
Deadenylation enzymes remove As from poly(A) tail
5’ de-capping
Internal cleavage of mRNA and fragment degradation
Post-translational Control - Protein
Degradation
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Proteins can be short-lived (e.g., steroid receptors) or
long-lived (e.g., lens proteins in your eyes).
Protein degradation in eukaryotes requires a protein cofactor called ubiquitin.

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Ubiquitin binds to proteins and identifies them for
degradation by proteolytic enzymes.
Amino acid at the N-terminus is correlated with protein
stability and determines rate of ubiquitin binding.
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Arg, Lys, Phe, Leu, Trp
Cys, Ala, Ser, Thr, Gly, Val, Pro, Met
1/2 life ≤3 minutes
1/2 life ≥ 20 hours
Epigenetics
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Non-sequence specific, heritable traits
Transcriptional gene silencing (TGS)
Imprinting
 X-inactivation
 RNA-induced transcriptional silencing (RITS)
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Post-transcriptional gene silencing (PTGS)
RNA-induced silencing complex (RISC)
 G quartets
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Post-translational protein-protein interactions
RNAi
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First described in C. elegans
Injecting antisense RNA into oocytes (ssRNA
that is complementary to mRNA)
Silences gene expression
Also injected dsRNA into oocytes found it was
10X more potent in inhibiting expression
Term RNAi
RNAi
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Higher eukaryotes produce a class of small RNA’s that
mediate silencing of some genes
Small RNA’s interact with mRNA in the 3’UTR and this
results in either mRNA degradation or translation
inhibition
Controls developmental timing in at least some
organisms
Used as a mechanism to protect against invading RNA
viruses (plants)
Controls the activity of transposons
Formation of heterochromatin
Mechanism of RNA Interference
Step 1
Step 2
Nat Rev Genet. 2002 Oct;3(10):737-47. Review.
Epigenetics



The study of mechanisms by which genes bring
about their phenotypic effects
Epigenetic changes influence Phenotype
without altering Genotype
Changes in properties of a cell that are inherited
but don’t represent a change in genetic
information.
A Histone Code?


Modifications of histones (usually the amino terminal
tails) convey epigenetic information
Types of modifications:

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Acetylation and methylation of lysine.
Methylation of arginine
Phosphorylation of serine
Ubiquitination of lysine.
The histone code hypothesis posits that serial
modifications provide a blueprint for reading chromatin
-for transcription, replication, repair, and
recombination.
Remodeling Nucleosomes


Changing the way nucleosomes bind to the DNA in
chromosomes is important to allow access to the
underlying DNA sequences during DNA replication,
repair, recombination, and transcription.
This occurs in three general ways:



Modification of the lys and Arg residues on the histone tails
decreases the grip of the nucleosome on DNA and causes
the nucleosome to slide more easily.
Variant histones are added to pre-existing nucleosomes
ATP-dependent protein “remodeling”complexes cause
nucleosomes to dissociate and/or slide along the DNA.