Lectures cover:
1. The Nature of Ribonucleic acid (RNA)
- 1 hour
2. Post-transcriptional Regulation
- 2 hours
Chapter 10
Nucleotides and Nucleic
Acids
Biochemistry
by
Reginald Garrett and Charles Grisham
Nucleotides and Nucleic Acids
• are biological molecules that possess heterocyclic nitrogenous
bases
• Nucleotide: see below
• Nucleic acid: linear polymer of nucleotides; serve a central
biological purpose – information transfer in cells
1.
2.
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA): serve in the expression of genetic
information stored in DNA through the processes of
transcription and translation
Importance of DNA and RNA
• Genetic information is stored in DNA
• Information encoded in a DNA molecule is
transcribed via synthesis of an RNA molecule
• The sequence of the RNA molecule is "read" and
is translated into the sequence of amino acids in
a protein
Information Transfer in Cells
Figure 10.1 The fundamental
process of information transfer
in cells. Information encoded
in the nucleotide sequence of
DNA is transcribed through
synthesis of an RNA molecule
whose sequence is dictated
by the DNA sequence. As the
sequence of this RNA is read
(as groups of three
consecutive nucleotides) by
the protein synthesis
machinery, it is translated into
the sequence of amino acids
in a protein. This information
transfer system is
encapsulated in the dogma:
DNA → RNA → protein.
What constitute nucleotide (nucleic acid)?
• Complete hydrolysis of nucleotide or nucleic acid
liberates:
1. nitrogenous base
2. five-carbon sugar
3. phosphoric acid
in equal amount
• A nucleotide is composed of a nitrogenous base,
a five-carbon sugar, and a phosphate
Nitrogenous base
• Two kinds of nitrogenous base are found in
nucleotides
1.
pyrimidines: six membered heterocyclic
aromatic ring containing two nitrogens
- two pyrimidines are found in RNA: cytosine and
uracil
2.
purines: 2 rings; one resembles pyrimindine ring
and the other resembles imidazole ring
- two purines are found in both RNA and DNA:
adenine and guanine
Figure 10.2 (a) The pyrimidine ring system; by convention, atoms are
numbered as indicated. (b) The purine ring system, atoms numbered as
shown.
Figure 10.3 The common pyrimidine bases—cytosine, uracil, and
thymine—in the tautomeric forms predominant at pH 7.
Figure 10.4 The common purine bases—adenine and guanine—in
the tautomeric forms predominant at pH 7.
The Sugar (Ribose)
• The sugar is pentose (five-carbon sugar)
• Ribonuceloside
- D-ribose → five-membered ring known as furanose
(D-ribofuranose)
• Deoxyribonucleoside
- 2-deoxy-D-ribose → five-membered ring (2-deoxyD-ribofuranose)
• Atoms are numbered as 1’, 2’, 3’
Figure 10.9 Furanose structures—ribose and deoxyribose.
What Is Nucleoside?
• A nucleoside = a nitrogenous base + a sugar
• Base is linked to sugar via a glycosidic bond
• Named by adding -osine to the root name of a purine
and -idine to the root name of a pyrimidine
Bases
Nucleosides
adenine
guanine
adenosine
guanosine
cytosine
uracil
+ Sugar
cytidine
uridine
Figure 10.10 b-Glycosidic bonds link
nitrogenous bases and sugars to form
nucleosides.
Figure 10.11 The common ribonucleosides—cytidine, uridine, adenosine, and
guanosine. Also, inosine drawn in anti conformation.
What Is Nucleotide?
• Nucleoside (base+sugar) + phosphate group
• Phosphoric acid is esterified to a sugar’s OH group
• C-5’ OH is esterified with phosphoric acid in the cell;
a ribonucleotide has a 5’ phosphate group
• The number of phosphate at the C-5’ can be 1, 2, or 3
Figure 10.13 Structures of the four common ribonucleotides—AMP, GMP,
CMP, and UMP—together with their two sets of full names, for example,
adenosine 5'-monophosphate and adenylic acid. Also shown is the
nucleoside 3'-AMP.
Figure 10.15 Formation of ADP and ATP by the successive addition of
phosphate groups via phosphoric anhydride linkages. Note the removal
of equivalents of H2O in these dehydration synthesis reactions.
Base
Nucleoside
Nucleotide
adenine
adenosine
adenosine 5’-monophosphate (AMP)
adenosine 5’-diphosphate (ADP)
adenosine 5’-triphosphate (ATP)
guanine
guanosine
GMP, GDP, GTP
cytosine
cytidine
CMP, CDP, CTP
uracial
uridine
UMP, UDP, UTP
What Is Nucleic Acid?
• Polymer of nucleotides linked 3’ to 5’ by
phosphodiester bridges – formed as nucleoside 5’monophosphate is successively added to the 3’ OH
group of the preceding nucleotide
• Polymer of ribonucleotides = RNA
• The convention way to read and write the
polynucleotide chain is from the 5’ end to the 3’ end
• Note that this reading actually passes through each
phosphodiester from 3’ to 5’
Figure 10.17
3'-5' phosphodiester bridges link nucleotides together to form polynucleotide chains.
TGCAT
Figure 10.18
Furanoses are represented by vertical lines; phosphodiesters are represented by
diagonal slashes in this shorthand notation for nucleic acid structures.
What Are the Different Classes of
Nucleic Acids?
• DNA - one type, one purpose
• RNA - 3 (or 4) types, 3 (or 4) purposes
– messenger RNA (mRNA)
– ribosomal RNA (rRNA)
– transfer RNA (tRNA)
– Small nuclear RNA (snRNA) - pre-mRNA splicing
– Small non-coding RNAs - post-transcriptional
gene silencing
Table 10-02, p.319
Messenger RNA (mRNA)
• Serves to carry information that is encoded
(stored) in DNA to the sites of protein synthesis
• In prokaryotes, a single mRNA contains the
information for synthesis of many proteins
• In eukaryotes, a single mRNA codes for just one
protein, but structure is composed of introns and
exons
Eukaryotic mRNA
• DNA is transcribed to produce heterogeneous
nuclear RNA
– mixed introns and exons with poly A
– intron - intervening sequence
– exon - coding sequence
– poly A tail - stability?
• Splicing produces final mRNA without introns
Ribosomal RNA (rRNA)
• Ribosomes are about 2/3 RNA, 1/3 protein
• rRNA serves as a scaffold for ribosomal
proteins
• Form complex secondary structures
• Their relative sizes are referred to as sedimentation
coefficients (S)
p.323
Figure 10.25 The organization and composition of prokaryotic and eukaryotic
ribosomes.
Transfer RNA tRNA)
•
•
•
•
Small polynucleotide chains - 73 to 94 residues each
Several bases usually methylated
Fold into a characteristic secondary structure
Each a.a. has at least one unique tRNA which carries
the a.a. to the ribosome
• 3'-terminal sequence is always CCA-a.a.
• Aminoacyl tRNA molecules are the substrates of
protein synthesis
p.325
DNA & RNA Differences?
Why is DNA 2'-deoxy and RNA is not?
• Vicinal -OH groups (2' and 3') in RNA make it
more susceptible to hydrolysis
• DNA, lacking 2'-OH is more stable
• This makes sense - the genetic material (DNA)
must be more stable
• RNA is designed to be used and then broken
down
Hydrolysis of Nucleic Acid
• RNA is resistant to dilute acid
• RNA is hydrolyzed by dilute base
• DNA and RNA are hydrolyzed by nucleases
Figure 10.29
The vicinal -OH groups of RNA
are susceptible to nucleophilic
attack leading to hydrolysis of
the phosphodiester bond and
fracture of the polynucleotide
chain; DNA lacks a 2'-OH
vicinal to its 3'-Ophosphodiester backbone.
Alkaline hydrolysis of RNA
results in the formation of a
mixture of 2'- and 3'-nucleoside
monophosphates.
Nucleases
• Enzymes that hydrolyze nucleic acids - DNase and RNase
• Nucleases are phosphodiesterase that cleave phosphodiester
bonds by using H2O
• Cleavage can be occurred on either side of the phosphorus; at
the 3’ side is labeled as “a” and at the 5’-side labeled as “b”
• Cleave internally is called endo; cleave from terminal
nucleotides is called exo
• Exo a: cleave at “a” from the 3’ end - snake venom
phosphodiesterase
• Exo b: cleave at “b” from the 5’ end – spleen
phosphodiesterase
Figure 10.30
Cleavage in
polynucleotide
chains: a
cleavage yields
5'-phosphate
products,
whereas b
cleavage gives
3'-phosphate
products.
Figure 10.31 Snake venom phosphodiesterase and spleen phosphodiesterase
are exonucleases that degrade polynucleotides from opposite ends.
•Pancreatic RNase A: an endo ribonuclease cleaves “b” after pyrimidines
(C or U)
Figure 10.32 An example of nuclease specificity: The specificity of RNA hydrolysis by
bovine pancreatic RNase. This RNase cleaves b at 3'-pyrimidines, yielding
oligonucleotides with pyrimidine 3'-PO4 ends.
Post-Transcriptional Regulation
• Biochemistry – Fourth Edition by Garrett &
Grisham
• Chapter 29 – Pages 974-981
Figure 29.1
Crick’s 1958 view of the
“central dogma of
molecular biology”:
Directional flow of
detailed sequence
information includes
DNADNA (replication),
DNA®RNA
(transcription),
RNAprotein
(translation), RNADNA
(reverse transcription).
Note that no pathway
exists for the flow of
information from
proteins to nucleic acids,
that is, proteinsRNA or
DNA. A possible path
from DNA to protein has
since been discounted.
Interestingly, in 1958,
mRNA had not yet been
discovered.
rRNAs constitute ~80-90% of total cellular RNAs
mRNAs only constitute ~3-5% of total cellular RNAs
What is post-transcriptional regulation ?
• Transcription: The process of RNA synthesis.
• Post-transcriptional regulation: The regulation
AFTER transcription, but not translation.
• AFTER DOES NOT mean that transcription has
to be complete.
How Are Eukaryotic Transcripts Processed and
Delivered to the Ribosomes for Translation?
• In prokaryotes, transcription and translation are concomitant
processes
• In eukaryotes, the two processes are spatially separated:
transcription occurs on DNA in the nucleus, and translation
occurs on ribosomes in the cytoplasm
• Thus, transcripts must be transported from the nucleus to the
cytosol to be translated
• On the way, these transcripts undergo processing
– Alterations that convert the newly synthesized RNAs
(primary transcripts) into mature mRNAs
• And unlike prokaryotes, eukaryotic mRNAs encode only one
polypeptide; i.e., they are monocistronic
Structural and spatial difference in gene expression
between procaryotes and eucaryotes
post-transcriptional
regulation
mRNA degradation
mRNA localization
Comparison between procaryotic
and eucaryotic mRNAs
polycistronic
monocistronic
Eukaryotic Genes are Split Genes
•
Introns intervene between exons
•
Exon: protein coding region; intron: noncoding region
•
Exon size is much smaller than intron size
•
Examples: actin gene has 309-bp intron separates first three amino
acids and the other 350 or so
•
Chicken pro-alpha-2 collagen gene is 40-kbp (40,000 bp) long, with 51
exons of only 5 kbp total; the exons range in size from 45 to 249 bases
•
Most introns (lengths vary from 60-10,000 bps) are untranslatable; they
need to be removed – splicing
•
Mechanism by which introns are excised and exons are spliced
together is complex and must be precise
Eukaryotic Genes are Split Genes
Figure 29.36 The organization of split eukaryotic genes.
The organization of the mammalian
dihydrofolate reductase (DHFR) gene
The gene is split into 6 exons spread over 31-kbp
The six exons are spliced together to give a 6-kb mRNA
Note that the size of the exons are much shorter than the introns,
and the exon pattern is more highly conserved than the intron pattern
Post-transcriptional regulation in the nucleus
Nuclear mRNA Processing Involves:
1.
2.
3.
Capping and Methylation
Polyadenylylation
Splicing
Capping and Methylation
• Primary transcripts (pre-mRNAs) are "capped“ as soon
as they are transcribed by RNA polymerase II
• The reaction is catalyzed by guanylyl transferase using
GTP as a substrate
• Capped G residue is methylated at N7-position
• Additional methylation occurs at C2'-O positions of
next two residues and at 6-amino group of the first
adenine (if A is the initial nucleotide)
The Capping of Eukaryotic pre-mRNAs
Figure 29.37
The capping of eukaryotic pre-mRNAs. Guanylyl transferase catalyzes the addition of a guanylyl
residue (Gp) derived from GTP to the 5-end of the growing transcript, which has a 5-triphosphate
group already there. In the process, pyrophosphate (pp) is liberated from GTP and the terminal
phosphate (p) is removed from the transcript. Gppp + pppApNpNpNp…  GpppApNpNpNp… + pp +
p (A is often the initial nucleotide in the primary transcript).
Figure 29.38
Methylation of several specific sites located at the 5-end of eukaryotic pre-mRNAs is an essential
step in mRNA maturation. A cap bearing only a single CH3 on the guanyl is termed cap 0. This
methylation occurs in all eukaryotic mRNAs. If a methyl is also added to the 2-O position of the first
nucleoside after the cap, a cap 1 structure is generated. This is the predominant cap form in all
multicellular eukaryotes. Some species add a third CH3 to the 2-O position of the second
nucleoside after the cap, giving a cap 2 structure. Also, if the first base after the cap is an adenine, it
may be methylated on its 6-NH2. In addition, approximately 0.1% of the adenine bases throughout
the mRNA of higher eukaryotes carry methylation on their 6-NH2 groups.
Cap 0
Cap 1
Cap 2
Enzymes involved in the Capping
Phosphatase
Guanylyl transferase
Guanine 7-Methyl transferase
2’ O-Methyl transferase
Why do cells need to cap their mRNA?
- cap is recognized by cap-binding proteins
- cap distinguishes mRNAs from other types of RNA molecules
(RNA pol I and III produce uncapped RNAs)
- mRNAs need a cap (and poly A tail) for export from the nucleus
- cap is necessary for translation
- cap stabilizes mRNA in the cytoplasm
3'-Polyadenylation and
transcriptional termination
3'-Polyadenylation
• Termination of transcription occurs only after RNA
polymerase has transcribed a consensus AAUAAA
sequence - the poly(A) signal
• 10-30 nucleotides after this site [the poly(A) signal], the
mRNA is cleaved and a string of ~200 adenine residues is
added to the mRNA transcript - the poly(A) tail
• poly(A) polymerase adds these A residues
• poly(A) tail bound by PABP stimulates translation and
governs the stability of mRNA
Signals required for the formation
of the 3’ end of mRNA
Figure 29.39
Poly(A) addition to the 3-ends of
transcripts occurs 10 to 35
nucleotides downstream from a
consensus AAUAAA sequence,
defined as the polyadenylylation
signal. CPSF (cleavage and
polyadenylylation specificity factor)
binds to this signal sequence and
mediates looping of the 3-end of
the transcript through interactions
with a G/U-rich sequence even
further downstream. Cleavage
factors (CFs) then bind and bring
about the endonucleolytic cleavage
of the transcript to create a new 3end 10 to 35 nucleotides
downstream from the
polyadenylylation signal. Poly(A)
polymerase (PAP) then
successively adds 200 to 250
adenylyl residues to the new 3end. (RNA polymerase II is also a
significant part of the
polyadenylylation complex at the
3-end of the transcript, but for
simplicity in illustration, its
presence is not shown in the lower
part of the figure.)
Polyadenylation of mRNA
A. Where is the template?
- does not require a template
- the poly(A) tail is not encoded in the genome
B. What’s the function of the polyA tail?
- by interaction with poly(A) binding protein (PABP),
it is necessary for efficient translation and protection
from mRNA degradation
Pre-mRNA splicing
splicing
translation
Nuclear Pre-mRNA Splicing
• Within the nucleus, pre-mRNA forms ribonucleoprotein
particles (RNPs) through association with a characteristic set
of nuclear proteins
• These proteins maintain the pre-mRNA in an untangled and
accessible conformation
• The substrate for splicing, that is, intron excision and exon
ligation, is the capped primary transcript emerging from the
RNA polymerase II transcriptional apparatus
• Splicing occurs exclusively in the nucleus
• Consensus sequences define the exon/intron junctions in
eukaryotic mRNA precursors
Splicing of Pre-mRNA
Capped, polyadenylated RNA, in the form of a RNP complex, is the
substrate for splicing
• In "splicing", the introns are excised and the exons are sewn
together to form mature mRNA
• The 5'-end of an intron in higher eukaryotes is always GU and the
3'-end is always AG
• All introns have a "branch site" 18 to 40 nucleotides upstream
from 3'-splice site
• The branch site is essential to splicing
What makes an intron?
5’ splice site
Branch site (usually
closer to 3’ss)
3’ splice site
R: Purine A or G
Y: Pyrimidine U or C
The Splicing Reaction Proceeds via
Formation of a Lariat Intermediate
• Next slide shows the splicing mechanism
• The branch site is usually YNYRAY, where Y = pyrimidine, R =
purine and N is anything
• The lariat a covalently closed loop of RNA is formed by
attachment of the 5'-P of the intron's invariant 5'-G to the 2'-OH
at the branch A site
• The exons then join, excising the lariat
• The lariat is unstable; the 2'-5' phosphodiester is quickly
cleaved and the intron is degraded in the nucleus
The Splicing Reaction Proceeds via
Formation of a Lariat Intermediate
Degraded
Splicing Depends on snRNPs
• Splicing depends on a unique set of small nuclear
ribonucleoprotein particles - snRNPs, pronounced
"snurps"
• A snRNP consists of a small RNA (100-200 bases long)
and about 10 different proteins
• Some of the 10 proteins are general, some are specific
(see Table 29.6)
• Major snRNP species are abundant, with more than
100,000 copies per nucleus
• snRNPs and pre-mRNA form the spliceosome
Splicing Depends on snRNPs
snRNPs Form the Spliceosome
• Splicing occurs when the various snRNPs come together with the
pre-mRNA to form a multicomponent complex called the
spliceosome
• The spliceosome is a large complex, about the size of a
ribosome; its assembly requires ATP
• snRNP U1 binds at the 5'-splice site, and U2 snRNP binds at the
branch site
• Interaction between the snRNPs brings 5'- and 3'- splice sites
together so lariat can form and exon ligation can occur
• The transesterification reactions that join the exons may in fact
be catalyzed by snRNAs themselves, but not by snRNP proteins
• Spliceosome assembly requires ATP-dependent RNA
rearrangements catalyzed by spliceosomal DEAD-box
ATPases/helicases
The spliceosome - RNA/protein complex
Composition:
Small nuclear RNAs (snRNAs):
5 snRNAs (U1, U2, U4, U5, U6), uridine rich, range in size from 106189 nucleotide long.
Proteins:
10 identified associated with each of the snRNAs to form the small
nuclear ribonucleoproteins (snRNPs)
non-snRNP splicing factors: SR-family proteins and other splicing
factors
(current estimate of total proteins is >50 different)
snRNPs Form the Spliceosome
1st event: U1 interacts with the 5’ splice site
Figure 29.43 Mammalian U1 snRNA can be arranged in a
secondary structure where its 5'-end is single-stranded and
can base-pair with the consensus 5'-splice site of the intron.
Assembly of
the spliceosome
Figure 29.44 Events in
spliceosome assembly. U1
snRNP binds at the 5'-splice
site, followed by the
association of U2 snRNP with
the UACUAA*C branch-point
sequence. The triple U4/U6U5 snRNP complex replaces
U1 at the 5'-splice site and
directs the juxtaposition of the
branch-point sequence with
the 5'-splice site, whereupon
U4 snRNP is released.
Constitutive Splicing vs. Alternative Splicing
Alternative Splicing Creates Protein Isoforms
• In constitutive splicing, every intron is removed and
every exon is incorporated into the mature RNA
• This produces a single form of mature mRNA from
the primary transcript
• However, many eukaryotic genes can give rise to
multiple forms of mature RNA transcripts
• This may occur by:
– Use of different promoters
– Selection of different polyadenylylation sites
– Alternative splicing of the primary transcript, or
– A combination of these three mechanisms
Constitutive mRNA Splicing
Every intron is removed and every exon is incorporated into mature RNA
without exception – results in a single form of mature mRNA
A cell
I
B cell
II
I
II
III
III
I
II
I
II
III
III
Alternative mRNA splicing I
Both isoforms are expressed in both A and B cell
A cell
I
B cell
II
I
I
III
II
III
III
I
II
I
I
III
II
III
III
Alternative mRNA splicing II
Different cell types express distinct isoform
A cell
I
B cell
II
I
II
III
III
I
C cell
II
I
I
III
II
III
III
I
II
I
III
III
Alternative mRNA Splicing Creates Protein Isoforms
•
•
•
•
•
•
Different transcript from a single gene make possible a set of related polypeptides,
termed protein isoforms, each with a slightly altered function
The isoforms of fast skeletal muscle troponin T are an example of alternative
splicing
This gene consists of 18 exons, 11 of which are found in all mature mRNAs and are
constitutive
Five of the exons (4 through 8) are combinatorial, in that they may be included or
excluded
Two (16 and 17) are mutually exclusive – one is always present but never both
64 different mature mRNA can be formed from this gene by alternative splicing
Alternative splicing expands the coding potential
of the genome – Tissue-specific splicing
mRNA Export
mRNA processing
7mG
AAAAAA
ppp
mRNA export
5’ end of mRNA leaves the nucleus first. Note exchange of some of
the proteins which do not leave the nucleus.
Post-transcriptional regulation
in the cytoplasm
Fates of mRNAs in the cytoplasm
• Translation
• Degradation
• Localization → translation
The competition between
mRNA translation and mRNA decay
Poly(A) nuclease:
removes poly(A) tail
- The mRNA is thought to be circularized by its interaction with eIF4E, eIF4G, and PABP
- This interaction stimulates mRNA translation and protects the 5’ and 3’ ends of the
mRNA from attack by decay enzymes
- Any factors affecting translation efficiency will have an opposite effect on its degradation
Two mechanisms of eucaryotic mRNA decay
General decay pathway: most mRNAs
Certain mRNAs: requires specific sequences
mRNA localization
Generally mRNAs are translated in the cytoplasm by free ribosomes;
their products may be directed to other sites in the cell
mRNAs encoding secreted or membrane-bound proteins are
directed to endoplasmic reticulum (ER) by a signal at the amino
terminus of the protein
Some mRNAs are directed to specific intracellular locations before
translation begins, i.e. translated at the site where the protein
functions
Basic features of mRNA localization include cis-acting elements
within the mRNA that targets the message to a subcellular region, a
protein-RNA complex that effects localization, and the cytoskeleton
that acts as a “road” for RNA movement
Most localization signal sequences appear to be in the 3'UTR
(untranslated region) of mRNAs
The importance of 3’ UTR in mRNA localization
5’UTR
Coding Region
3’UTR
5’UTR
Coding Region
mRNA encoding hairy protein are
normally localized to the apical
site of nuclei
Injected hairy RNA containing the 3’ UTR
Injected hairy RNA lacking the 3’ UTR
RNA Editing: Another Way To Increase the
Diversity of Genetic Information
• RNA editing is a process that changes one or more nucleotides
in an RNA transcript by deaminating a base, either A→I or C→U
• These changes alter the coding possibilities in a transcript,
because I will pair with G (not U as A does) and U will pair with
A (not G as C does)
• RNA editing can increase protein diversity by
(1) altering amino acid coding possibilities
(2) introducing a premature stop codon
(3) changing splice site in a transcript
RNA editing – A to I editing
A ----- U
I ----- C
Carried out by ADAR- adenosine deaminase acting on RNA, which recognizes
a double-stranded RNA region
Such regions form when an exon region containing the A to be edited base pairs with a
complementary base sequences in an intron known as the editing site complementary
sequence
Editing of apolipoprotein B (apoB) mRNA – C to U
Non-edited mRNA encodes 550-kDa protein;
functions to make liver-derived VLDL
Carried out on a single-stranded regions of
transcript by an editosome whose core structure
consists of a cytosine deaminase and an adapter
protein that brings the deaminase and the transcript
together
Editing of CAA codon to UAA stop site at codon
2152; edited mRNA encodes 250-kDa protein;
functions to make intestinal-derived lipid
complexes
Unified theory of gene expression
Stages: transcription → transcript processing → mRNA export → translation
Traditionally they have been presented as a linear series of events
(a pathway of discrete and independent steps) – each going to completion
before the next begins
Now it is clear that each stage is part of a continuous process with
physical and functional connections between the transcriptional and
processing machineries.
Capping, RNA splicing, 3’ end formation and polyadenylation, and nuclear
export are coupled to transcriptional machinery
Regulation occurs at multiple levels in this continuous process
in a coordinated fashion
Eucaryotic cells have elaborate mRNA surveillance systems to destroy any
messages containing errors