Chap. 4. Basic Molecular Genetic
Mechanisms (Part A)
Topics
• Structure of Nucleic Acids
• Transcription of Protein-coding Genes and Formation of
Functional mRNA
• Decoding of mRNA by tRNAs
• Stepwise Synthesis of Proteins on Ribosomes
• DNA Replication
Goals
To learn the basic
mechanisms of
transcription, RNA
processing,
translation, and
replication
Fig. 4.1.
The “Incredible Hulk of Hounds”
The “double
muscle” phenotype
commonly occurs
due to defective
splicing of the
myostatin gene in
animals. Myostatin
is a protein that
limits skeletal
muscle
development.
Nucleic Acid Strands
Nucleic acids are polymers derived
from nucleotides. As shown in Fig.
4.2a, the backbone consists of
repetitive [-phosphate-(deoxy)ribose-]
units. Individual nucleotides are joined
by phosphodiester linkages. Like
proteins, nucleic acid chains have a
polarity which is defined by the 5' and
3' ends of the sequence. By
convention, the sequence of bases in a
strand is written left-to-right from
the 5' to 3' end (Fig. 4.2b).
Structure of Double-helical DNA
Cellular DNA exists
primarily in a right-handed
double-helical form (Fig.
4.3). The double helix
contains two interwound,
antiparallel DNA strands
(see arrows). The strands
are complementary and pair
together via Watson-Crick
base pairs (A.T; G.C). The
backbones of the strands
are located on the outside
of the helix, while the
bases are stacked inside.
In the most common conformation of the double helix (B DNA),
~10 bp occur per turn. B DNA molecules contain wide (major)
and narrow (minor) grooves in which parts of the bases are
exposed to the outside. DNA binding proteins locate and
interact with specific base sequences exposed in the grooves.
DNA Bending
DNA can be bent because it lacks stabilizing bonds oriented
parallel to the axis of the double helix. Bending commonly
occurs on binding of transcription factors, e.g., TATA boxbinding protein (TBP) (Fig. 4.5), and DNA-binding proteins such
as histones. Bending is crucial for the packaging of DNA in
chromatin inside eukaryotic cells.
Supramolecular Structure
In many cases, multimeric proteins
achieve extremely large sizes,
e.g., 10s-100s of subunits. Such
complexes exhibit the highest level
of structural organization known as
supramolecular structure. Examples
include mRNA transcription
preinitiation complexes (Fig. 3.12),
ribosomes, proteasomes, and
spliceosomes. Typically,
supramolecular complexes function
as ”macromolecular machines" in
reference to the fact that the
activities of individual subunits are
coordinated in the performance of
some overall task (e.g., protein
synthesis by the ribosome).
Base-catalyzed Hydrolysis of RNA
DNA and RNA differ w.r.t. the sugar found in their nucleotide
monomer units (2-deoxyribose vs ribose). The phosphodiester
bonds of RNA are susceptible to hydrolysis in basic solution due
to the presence of the 2’-hydroxyl group of ribose (Fig. 4.6).
In contrast, the phosphodiester bonds in DNA are much less
susceptible because 2-deoxyribose lacks this group. It is
thought that DNA was selected over RNA as the preferred
molecule for long-term storage of genetic information because it
is a less reactive molecule due to its containing 2-deoxyribose.
DNA Denaturation/Renaturation
DNA denaturation refers to the unwinding and separation (melting)
of the two strands of a double-helical DNA molecule. In vivo,
enzymes such as helicases, for example, separate DNA strands. In
vitro, denaturation is achieved by heating (Fig. 4.7a) or by
treatment with low ionic strength buffers or extremes of pH. DNA
denaturation can be monitored by UV absorption spectroscopy.
Because G.C base pairs are held together by 3 H-bonds, whereas
A.T base pairs are connected by 2 H-bonds, the melting point of
double helical DNA increases with G+C content (Fig. 4.7b). In
DNA renaturation, double helical molecules are formed by the
annealing of single-stranded molecules.
DNA Supercoiling
Processes such as replication that unwind double-helical DNA
introduce torsional stress that results in supercoiling. This is
most evident in circular DNA molecules such as bacterial plasmids
and some viruses (Fig. 4.8a), but occurs in linear eukaryotic
chromosomes as well. Replication causes over-winding ahead of
the strand separation site and positive supercoiling. Underwinding can occur resulting in negative supercoiling. Enzymes
called topoisomerases regulate the amount of supercoiling in DNA
in vivo. Topoisomerase I relaxes supercoiled DNA (Fig. 4.8b).
Topoisomerase II introduces negative supercoils into DNA.
Cellular DNA naturally exists in a slightly negatively supercoiled
state. Negative supercoiling facilitates processes such as
replication and transcription, where DNA is unwound.
Nick
RNA Structure
Most cellular RNAs consist of a single strand. However, doublehelical regions are common in RNA where complementary sequence
regions occur. Common types of RNA secondary structure elements
are hairpins and stem-loop structures (Fig. 4.9a). Stem regions
form A DNA-type double helices. The turns connecting the helices
are shorter in hairpins than in stem-loops. RNAs also form
elaborate 3D structures in molecules such as tRNA and rRNA. The
”pseudoknot” tertiary structure found in the human telomerase
RNA is illustrated in Fig. 4.9b. The 3D structures of ribozymes
rival those of proteins with respect to complexity.
Mechanism of Transcription
In transcription, a sequence in
DNA is copied into RNA in a
reaction catalyzed by RNA
polymerase. The RNA
synthesized is complementary to,
and runs antiparallel to the
template strand (Fig. 4.10a).
The RNA sequence is the same
as the coding strand (not shown).
Growth of the RNA chain occurs
in the 5' to 3' direction. The
mechanism of the nucleotide
polymerization reaction proceeds
with the elimination of inorganic
pyrophosphate (PPi). The
transcription start site is
numbered +1. DNA sequences
preceding the start site are
located "upstream" whereas
sequences after the start site
are located "downstream."
Conventions for Describing RNA
Transcription
The primary RNA transcript produced in transcription has the same
sequence as the coding or nontemplate strand of DNA (Fig. 4.10b).
It is complementary to the template strand of DNA. The promoter
is a DNA sequence in the nontemplate strand of DNA that tells
RNA polymerase where to begin transcription (+1 site) of a gene.
Transcription Initiation
Transcription by RNA polymerase occurs in 3 stages-initiation, elongation, and termination (Fig. 4.11). In initiation,
RNA polymerase binds to a promoter, typically with the
assistance of transcription factors. About 14 base pairs of
DNA are melted (the transcription bubble), and the enzyme
synthesizes a 2-nucleotide RNA complementary to the template
strand. Transcription is regulated primarily at the initiation
stage.
Transcription Elongation
After initiation, RNA polymerase clears the promoter, leaving
transcription factors behind. It maintains a 14-residue bubble
as it moves downstream elongating the RNA, which grows in the
5' to 3' direction. The nascent RNA remains H-bonded to the
template strand via its last 8 nucleotides (the DNA-RNA hybrid
region). Elongation proceeds at ~1,000 nucleotides per minute in
eukaryotes.
Transcription Termination
When RNA polymerase encounters a termination sequence,
transcription stops and the polymerase and the completed
"primary transcript" are released from the DNA. RNA polymerase
then is free to initiate transcription at another promoter.
Gene Organization in Prokaryotes
About half of the genes in
prokaryotic cells occur in
transcription units known
as operons. Operons are
transcribed from a single
promoter and usually
contain genes that
participate in a common
process such as synthesis
of tryptophan, e.g., the
trp operon of E. coli (Fig.
4.13a). The trp operon
mRNA is polycistronic and
encodes 5 different
proteins. Each cistron
coding sequence is
translated into a protein.
Gene Organization in Eukaryotes
Most eukaryotic genes are
transcribed separately,
even in simple organisms
such as yeast. The TRP
biosynthesis genes, for
example, each have their
own promoter and actually
are encoded on different
chromosomes in yeast
(Fig. 4.13b). In addition,
gene coding sequences in
higher eukaryotes
typically are interrupted
with non-translated
sequences known as
introns. Intron sequences
in pre-RNA are removed
by RNA processing
reactions prior to
formation of the final
functional mRNA.
Eukaryotic pre-mRNA Processing: Capping
Bacterial mRNAs are functionally active
as transcribed. Eukaryotic pre-mRNAs
must be extensively processed to attain
their final functional forms. The
modification that occurs at the 5' end
of the primary transcript is called the
5' cap (m7Gppp) (Fig. 4.14). In this
modification, a 7-methylguanylate
residue is attached to the first
nucleotide of the pre-mRNA by a 5'-5'
linkage. The 2'-hydroxyl groups of the
ribose residues of the first 2
nucleotides may also be methylated. The
5' cap is important for transport of the
mRNA to the cytoplasm, protection
against nuclease degradation, and
initiation of translation.
Eukaryotic pre-mRNA Processing: Splicing
& Polyadenylation
Eukaryotic pre-mRNAs are
capped at the 5' end while
being transcribed. They also
are modified at the 3' end by
polyadenylation (Fig. 4.15).
This involves cleavage of the
longer pre-mRNA at the
polyadenylation site and the
addition of up to 250
adenylate residues by
template-independent poly(A)
polymerase. Non-coding RNA
intron sequences are excised
and the coding exon sequences
are ligated to form the
functional mRNA by the
process known as splicing. The
mRNA retains 5' and 3'
untranslated regions (UTRs)
at each end.The poly(A) tail
helps protect the mRNA from
nuclease digestion.
The “Incredible Hulk of Hounds”
The “double
muscle” phenotype
commonly occurs
due to defective
splicing of the
myostatin gene in
animals. Myostatin
is a protein that
limits skeletal
muscle
development.
Alternative Splicing & Gene Regulation
Protein domains can be encoded by a single exon or by a small
collection of exons within a larger gene. The coding regions for
domains can be spliced in or out of the primary transcript by the
process of alternative splicing. The resulting mRNAs encode
different forms of the protein, known as isoforms. Alternative
splicing is an important method for regulation of gene expression
in different tissues and different physiological states. It is
estimated that 60% of all human genes are expressed as
alternatively spliced mRNAs. Alternative splicing is illustrated in
Fig. 4.16 for the fibronectin gene. The fibroblast and hepatocyte
isoforms differ in their content of the EIIIA and EIIIB domains
which mediate cell surface binding.Twenty different isoforms of
fibronectin produced by alternative splicing have been identified.