Download Freeman 1e: How we got there

CHAPTER 7
Essentials of Molecular Biology
Genes and Gene Expression
Informational macromolecules = DNA, RNA, Protein
Unit of information = gene (a segment of DNA specifying a protein, rRNA or tRNA)
Genes = coded by DNA or RNA (HIV)
• The three key processes of macromolecular
synthesis are: 1. DNA replication;
•2. transcription (the synthesis of RNA from a
DNA template); and
•3. translation (the synthesis of proteins using
messenger RNA as a template).
• Although the basic processes are the same in
prokaryotes and eukaryotes, the organization of
genetic information is more complex in eukaryotes.
• Most eukaryotic genes have both coding
regions (exons) and noncoding regions
(introns). Both introns and exons are
transcribed into the primary transcript, an
unprocessed RNA molecule that is the direct
product of transcription.
DNA Structure: The Double Helix
• DNA is a double-stranded molecule that forms a helical
configuration and is measured in terms of numbers of base
pairs.
•Double stranded molecule is arranged in an antiparallel
fashion.
Purine = AG
Pyrimidines = CT
• The two strands in the double helix are antiparallel.
DNA size is expressed as bp, kbp or
Mbp
Size of E. coli DNA = 4640 kbp or
4.64 Mbp
Inverted Repeats, Secondary
Structure, and Stem-Loops
Inverted repeats allow for the formation of secondary structure.
Secondary structure, stem-loop
is more common in RNA than
in DNA
• The strands of a double-helical DNA molecule can
be denatured by heat and allowed to reassociate
following cooling (annealing and hybridization).
sDNA absorbed more uv
than dDNA
• In all cells, DNA exists as two
polynucleotide strands whose base sequences
are complementary.
•The complementarity of DNA arises from the
specific pairing of the purine (AG) and
pyrimidine (CT) bases.
•Adenine always pairs with thymine, and
guanine always pairs with cytosine.
DNA Structure: Supercoiling
• The very long DNA molecule can be packaged into
the cell because it is supercoiled (Figure 7.8).
A break in a phophodiester bond
(nick) changes a supercoiled
molecule to a relaxed molecule
• In prokaryotes, this supercoiling is produced by
enzymes called topoisomerases. In eukaryotic
chromosomes, DNA is wound around proteins called
histones, forming structures called nucleosomes.
• Topoisomerases - DNA gyrase is a key enzyme in
prokaryotes, introducing negative supercoils to the DNA
(Figure 7.10). Reverse gyrase introduces positive
supercoiling.
Chromosomes and Other
Genetic Elements
• In addition to the chromosome, a number
of other genetic elements exist in cells.
• Plasmids are DNA molecules that exist
separately from the chromosome of the cell.
Mitochondria and chloroplasts contain their
own DNA chromosomes.
•Viruses contain a genome, either DNA or
RNA, that controls their own replication.
Transposable elements exist as a part of other
genetic elements.
• Table 7.2 shows the number, size, and configuration
of chromosomes in a few microorganisms, both
prokaryotic and eukaryotic.
DNA Replication
• Both strands of the DNA helix serve as templates for
the synthesis of two new strands (semiconservative
replication).
• The two progeny double helices each contain one
parental strand and one new strand. The new strands
are elongated by addition to the 3' end.
5’ (PO4)
3’ (OH)
• DNA polymerases require a primer, which
is composed of RNA (Figure 7.13).
E. coli polymerases = pol I-V
Pol III is the primary enzyme for DNA synthesis
It has 3 activities – 5’-3’ synthesis; 5’-3’ exo and 3’-5’ exonuclease
Pol I has 5’-3’ synthesis and 5’-3’ exo (to remove RNA primers)
DNA Replication: The
Replication Fork
• In prokaryotes, DNA synthesis begins at a unique
location called the origin of replication.
• Table 7.3 shows the major enzymes
involved in DNA replication in Bacteria. The
double helix is unwound by helicase and is
stabilized by single-strand binding protein.
• As replication proceeds, the site of replication, called
the replication fork, appears to move down the DNA.
Okazaki fragment
• Extension of the DNA occurs continuously on the
leading strand but discontinuously on the lagging
strand (Figure 7.15).
Sealing of nicks at the lagging strand
Errors in base pairing are corrected by proofreading functions associated
with the activities of DNA polymerases.
Pol I and III have 3’-5’ exonuclease that removes mismatched nucleotide
Function of 5’-3’ activity?
Replication of prokaryotic chromosome
• In Escherichia coli, and probably in all prokaryotes
that contain a circular chromosome, replication is
bidirectional from the origin of replication.
Replisome
Tools for Manipulating DNA
Restriction Enzymes and
Hybridization
• Restriction enzymes recognize specific short sequences in DNA and
make breaks in the DNA.
•EcoR1 cuts only unmodified DNA
modified by EcoR1 methylase
Palindrome
• The products of restriction enzyme digestion can be
separated using gel electrophoresis
Gel electrophoresis
Nomenclature
• The Southern blot (hybridization) technique is used
to hybridize probes to DNA fragments that have
been separated by gel electrophoresis to identify
complementary sequences.
Fragments complementary
to the probe are circled
yellow on the separation gel
which hybridized to the
probe.
Sequencing and Synthesizing DNA
• DNA can be sequenced by the Sanger method,
which involves copying the DNA to be sequenced in
the presence of chain-terminating dideoxynucleotides
• The final products are separated by electrophoresis and the
sequence is read. The short DNA primers required in this
method can be synthesized chemically.
Sequencing Methods Sanger
method –(enzymatic, dideoxy chain
termination)
Dye-termination sequencing. This
is a much more versatile method of
sequencing, because it is not
necessary to have a chemically
modified oligonucleotide. The
fluorescent dyes are conjugated to
dideoxynucleotides, so a chain
termination event is marked with a
unique chemical group. Only one
reaction needs to be run in this case,
because there is no longer a
separation between the label and the
terminating group.
Maxum and Gilbert method –
(chemical degradation)
Synthesis of nucleotides
for primers, probes and
Site-directed mutagenesis
Solid-phase procedure – First
nucleotide is fastened to an
insoluble porous support (50µ m
silica gel)
Amplifying DNA: The Polymerase
Chain Reaction
• The polymerase chain reaction (PCR) is a
procedure for amplifying DNA in vitro and employs a
heat-stable DNA polymerase from thermophilic
prokaryotes.
Heat (95oC) is used to denature
the DNA into two single-stranded
molecules, annealing of primers is
achieved by reducing temp (70oC)
and DNA synthesis (Primer
extension) at (50-60oC) in which
each of the strand is copied by the
polymerase. After each cycle, the
newly formed double strands are
again separated by heat, and a new
round of copying proceeds. At
each cycle, the amount of target
DNA doubles.
Annealing
•Applications of PCR
•PCR is a extremely sensitive and specific and
highly efficient method
•Used in identifying organisms – 16 sRNA analysis
•Clinical diagnostics – to identify infectious agents
•DNA fingerprinting – in forensic analysis to
identify individuals
•Gene expression studies – RT-PCR
RNA Synthesis: Transcription,
• The three major types of RNA are
messenger RNA (mRNA), transfer RNA
(tRNA), and ribosomal RNA (rRNA).
• Transcription of RNA from DNA involves
the enzyme RNA polymerase, which adds
bases onto the 3' ends of growing chains.
Unlike DNA polymerase, RNA polymerase
needs no primer and recognizes a specific
start site on the DNA called the promoter.
Transcription
β, β’, α2 - coreenzyme
β, β’, α2, σ – holoenzyme
EM
Diversity of Sigma Factors,
Consensus Sequences, and
Other RNA Polymerases
• In Bacteria, promoters are recognized by the sigma
subunit of RNA polymerase. Promoters recognized
by a specific sigma factor have very similar
sequences.
σ 70 is the major sigma factor in E. coli
Heat-shock sigma factor
Nitrate – dependent “
Flagella- specific gene “
Figure 7.30 shows the sequence of a few promoters
from Escherichia coli.
• In the Eukarya, the major classes of RNA are transcribed by different RNA
polymerases, with RNA polymerase II producing most mRNA.
RNA pol I – most rRNA
RNA pol II – all mRNA
RNA pol III –tRNA and one type of rRNA
•The single RNA polymerase of Archaea resembles RNA polymerase II in both
structure and function.
INR = Initiator element
Transcription Terminators
• RNA polymerase stops transcription at specific sites
called transcription terminators (Figure 7.32).
Rho dependent – rho
causes termination by
binding to mRNA
Intrinsic terminators –
stem and loop
structure with specific
sequences poly U at
3’ and at stem.
• Although encoded by DNA, these signals
function at the level of RNA. Some are
intrinsic terminators and require no accessory
proteins beyond the polymerase. In Bacteria,
these sequences are often stem-loops followed
by a run of U's. Other terminators require
proteins, such as Rho.
The Unit of Transcription
• Moncistronic vs. polycistronic mRNA
•The unit of transcription often contains more
than a single gene. Transcription of several
genes into a single mRNA molecule may
occur in prokaryotes, and so the mRNA may
contain the information for more than one
polypeptide (Figure 7.33).
• Genes that are transcribed together from a
single promoter constitute an operon. In all
organisms, genes encoding rRNA are
cotranscribed but then are processed to form
the final rRNA species.
Protein Synthesis - The Genetic Code
• The genetic code is expressed in terms of RNA, and
a single amino acid may be encoded by several
different but related codons.
• Table 7.5 shows the genetic code as expressed by
triplet base sequences of mRNA. A codon is
recognized following specific base-pairing with a
sequence of three bases on a tRNA called the
anticodon.
• Some tRNAs can recognize more than one
codon. In these cases, tRNA molecules form
standard base pairs only at the first two
positions of the codon, while tolerating
irregular base pairing at the third position.
This apparent mismatch phenomenon is called
wobble (Figure 7.34).
• A few codons, called nonsense codons, do
not encode an amino acid. In addition to the
nonsense codons, there is also a specific start
codon that signals where the translation
process should begin.
• It is important to have a precise starting
point because with a triplet code, it is critical
that translation begin at the correct location. If
it does not, the whole reading frame will be
shifted and an entirely different protein (or no
protein at all) will be formed (Figure 7.35).
Transfer RNA
• One or more transfer RNAs (Figure 7.36)
exist for each amino acid found in a protein.
Enzymes called aminoacyl-tRNA
synthetases (Figure 7.37) attach an amino
acid to a tRNA.
Aminoacyl tRNA synthetases (one for tRNAs
of each a. a.)
• Once the correct amino acid is attached to its
tRNA, further specificity resides primarily in
the codon-anticodon interaction.
Translation: The Process of
Protein Synthesis
• The ribosome plays a key role in the
translation process, bringing together mRNA
and aminoacyl tRNAs.
Shine-Dalgarno
sequence (3-9
nucleotides)
Formylmethionine
tRNA – specific for
start codon, AUG
• There are three sites on the ribosome: the
acceptor site, where the charged tRNA first
combines; the peptide site, where the growing
polypeptide chain is held; and an exit site.
• During each step of amino acid addition, the
ribosome advances three nucleotides (one
codon) along the mRNA, and the tRNA
moves from the acceptor to the peptide site.
Termination of protein synthesis occurs when
a nonsense codon, which does not encode an
amino acid, is reached.
• During each step of amino acid addition, the
ribosome advances three nucleotides (one
codon) along the mRNA, and the tRNA
moves from the acceptor to the peptide site.
Termination of protein synthesis occurs when
a nonsense codon, which does not encode an
amino acid, is reached.
• Several ribosomes can translate a single
mRNA molecule simultaneously, forming a
complex called a polysome.
Folding and Secreting Proteins
• To function correctly, proteins must be
properly folded. Folding may occur
spontaneously but may also involve other
proteins called molecular chaperones
(Figure 7.40).
Molecular chaprones
Two systems in E. coli
• Many proteins also must be transported into
or through cell membranes. Such proteins are
synthesized with a signal sequence (Figure
7.41) that is recognized by the cellular export
apparatus and is removed either during or
after export.