Download Introduction and Review

Jerald D. Hendrix
Last Updated: Summer 2016
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Cell Architecture
Review of Cell Chemistry
How Cells are Studied
Transcription,
RNA Processing, and Transcriptional Regulation
Translation and the Genetic Code
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Alberts, Panel 1-2
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Panel 2–1 Chemical bonds and groups
Panel 2–2 The chemical properties of water
Panel 2–3 An outline of some of the types of sugar
Panel 2–4 Fatty acids and other lipids
Panel 2–5 The 20 amino acids found in proteins
Panel 2–6 A survey of the nucleotides
Panel 2–7 The principal types of weak noncovalent bonds
Panel 3–1 Free energy and biological reactions
Panel 4–1 A few examples of some general proteins
Panel 4–2 Four different ways of depicting a small protein
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Visualization Approach
Microscopy
Alberts Panel 1-1
 X-ray diffraction crystallography
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Biochemistry Approach
Making Antibodies
Alberts Panel 4-3
 Fractionation
Alberts Panel 4-4
 Chromatography
Alberts Panel 4-6
 Electrophoresis
Alberts Panel 4-7
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
Genetic Approach
A.
B.
C.
D.
E.
F.
G.
Structure of RNA
Major Classes of RNA
Transcription in Prokaryotes
Transcription in Eukaryotes
Post-transcriptional Processing of Eukaryotic mRNA
Transcriptional Regulation in Prokaryotes:
the Lac Operon as an example
Transcriptional Regulation in Eukaryotes:
Steroid Hormones as an Example
1.
2.
3.
4.
Uracil instead of Thymine
Ribose instead of Deoxyribose
Usually single-stranded
May have hairpin loops (e.g. loops in tRNA)
Messenger RNA
1.
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
mRNA
Contains information for the amino acid sequences of proteins
Transfer RNA
2.
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
tRNA
Attaches to an amino acid molecule and interfaces with mRNA during
translation
Ribosomal RNA
3.
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
rRNA
Structural component of ribosomes
Small nuclear RNA
4.
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

snRNA
Component of small ribonucleoprotein particles
Processing of mRNA
Small nucleolar RNA
5.
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
snoRNA
Processing of rRNA
Small cytoplasmic RNAs
6.

Variable functions; many are unknown
Micro RNA
7.
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miRNA
Inhibits translation of mRNA
Small interfering RNA
8.
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
siRNA
Triggers degradation of other RNA molecules
Piwi-interacting RNA
9.
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
piRNA
Thought to regulate gametogenesis
Requires a double-stranded DNA template
1.
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The DNA strands separate, and only one of the strands is used as a
template for transcription
“Template strand” and “nontemplate strand”
Direction and numbering conventions
2.
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From the 3’  5’ direction on the template strand is called
“downstream”
From the 5’  3’ direction on the template strand is called “upstream”
The nucleotide at the transcriptional start site is designated “+1” and the
numbering continues +2, +3, etc. in the downstream direction
The nucleotide immediately upstream from +1 is designated “-1” (there
is no 0); numbering continues -1, -2, etc. in the upstream direction
3.
4.
5.
Transcription requires nucleoside triphosphates (NTPs; ATP,
GTP, CTP, UTP) as raw materials
Nascent RNA strand synthesis (elongation) occurs only in the 5’
 3’ direction, with new nucleotides added to the 3’ end of the
nascent strand
Transcription is catalyzed by DNA-directed RNA polymerases
The initiation of transcription occurs when RNA polymerase
binds to a “promoter region” upstream from the transcriptional
start site
Promoter regions typically have short stretches of common
nucleotide sequences, found in most promoters, called
“consensus sequences”
Common prokaryotic (bacterial) consensus sequences include:
6.
7.
8.
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-10 consensus sequence: TATAAT box or Pribnow box
-35 consensus sequence: TTGACA
-40 to -60: Upstream element; repetitive A-T pairs
Bacterial RNA polymerase consists of a core enzyme and a
sigma factor
Bacterial RNA polymerase core has 4 or 5 subunits
9.
10.
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11.
α2ββ‘ω
α2ββ‘ is essential; ω is not
Sigma factors (σ) are global regulatory units. Most bacteria
possess several different sigma factors, each of which mediate
transcription from several hundred genes …
… for example:
11.
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12.
In E. coli, during log (exponential) growth, the major sigma factor
present is σ70
During stationary phase, it is σS
Shifting from σ70 to σS activates the transcription of multiple genes
linked to survival during stationary phase
Transcription begins when the core RNA polymerase attaches to
a sigma factor to form a holoenzyme molecule
13.
14.
15.
16.
The holoenzyme binds to a promoter, and the dsDNA template
begins to unwind
A nascent RNA strand is started at +1 on the template
After transcription is initiated, the sigma factor often dissociates
from the holoenzyme
RNA polymerase moves 3’  5’ along the template,
synthesizing the nascent RNA
5’  3’
17.
18.
19.
Transcription ends (termination) when RNA polymerase
reaches a terminator sequence, usually located several bases
upstream from where transcription actually stops
Some terminators require a termination factor protein called the
rho factor (); these are rho-dependent. Others are rhoindependent.
Messenger RNA in bacteria is often polycistronic, which means
that it has the code for >1 protein on a single mRNA molecule;
mRNA in eukaryotes is almost always monocistronic
1.
2.
Chromatin in eukaryotes is unfolded to permit access to the
template DNA during transcription
Eukaryotic promoters
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Recognized by accessory proteins that recruit different RNA polymerases
(I, II, or III)
Consist of a core promoter region and a regulatory promoter region
Core promoter region is immediately upstream from the coding region
Usually contains:
TATA box – Consensus sequence at -25 to -30
and other core consensus sequences
2.
…

Regulatory promoter region
Immediately upstream from the core promoter, from about -40 to -150
Consensus sequences include:
OCT box
GC box
CAAT box
3.
Eukaryotic RNA polymerases
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RNA polymerase I: Synthesizes pre-rRNA
RNA polymerase II: Synthesizes pre-mRNA
RNA polymerase III: Synthesizes tRNA, 5S rRNA, and several small
nuclear and cytosol RNAs
Also, the different RNA polymerases use different mechanisms for
termination
1.
2.
In eukaryotes, mRNA is initially transcribed as precursor
mRNA (“pre-mRNA”). This is part of a transcript called
heterogeneous nuclear RNA (hnRNA); the terms hnRNA and
pre-mRNA are sometimes used interchangably.
Almost all eukaryotic genes contain introns: noncoding regions
that must be removed from the pre-mRNA. The coding regions
are called exons.
3.
4.
5.
Introns are removed, and the exons are spliced together, by
ribonucleoprotein particles called spliceosomes.
mRNA contains a “leader sequence” at its 5’ end, before the
coding region. The coding region begins with a translational
initiation codon (AUG).
A methylated guanosine cap is added to the 5’ end of the
mRNA by capping enzymes. The cap is attached by a 5’  5’
triphosphate linkage
6.
7.
8.
The coding region ends with one or more translational
termination codons (stop codons).
At the 3’ end is a noncoding trailer region.
A 3’ poly-A tail, consisting of 50 – 250 adenosine nucleotides, is
added to the 3’ end by a 3’ terminal transferase enzyme.
1.
2.
Operon: A group of genes in bacteria that are transcribed and
regulated from a single promoter
Constitutive vs. regulated gene expression
Constitutive gene expression: When a gene is always transcribed
 Regulated gene expression: When a gene is only transcribed under
certain conditions
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3.
The lac operon in E. coli consists of:

3 structural genes (genes that encode mRNA)
 lac z gene: Encodes β-galactosidase
 lac y gene: Encodes β-galactoside permease
 lac a gene: Encodes β-galactoside transacetylase
The lac promoter gene: lac p
 The lac repressor gene: lac i (constitutively expressed and transcribed
from its own promoter, different from lac p)
 The lac operator region: lac o (which overlaps lac p and lac z)
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4.
The genes of the lac operon are only transcribed in the presence
of lactose (or another chemically similar inducer)
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In the absence of lactose, the lac repressor protein binds to lac o (lac
operator) and blocks RNA polymerase from binding to the promoter (lac
p)
In the presence of lactose:
 Lactose in the cell is converted to allolactose
 Allolactose binds to the lac repressor protein, causing it to causing it to
dissociate from the operator so RNA polymerase can reach the promoter
5.
Transcription of the lac operon is stimulated by conditions of
low glucose concentration

When glucose levels are low:
 Adenylate cyclase activity is high and the concentration of cyclic AMP
(cAMP) is high
 cAMP binds to the catabolite activator protein (CAP)
 The cAMP/CAP complex increases the efficiency of binding of RNA
polymerase to the promoter
 So there is increased lac transcription
…
5.
When glucose levels are high:
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6.
Adenylate cyclase activity is lowered, so cAMP levels are low
This means there is much less cAMP/CAP complex
And there is decreased lac transcription
So … E. coli will metabolize glucose first, then lactose when the
glucose runs out
1.
2.
3.
4.
Steroid hormones are secreted by endocrine gland cells and
travel through the bloodstream
The steroid enters the cytoplasm of target cells and binds to a
cytoplasmic steroid receptor protein
The steroid receptor/steroid complex enters the nucleus, where
it binds to regulatory sites (typically upstream from specific
promoters)
Transcription from some promoters may be activated (“turned
on”) while transcription from other promoters may be inhibited
(“turned off”)
5.
6.
Once the genes that have been activated by the steroid
receptor/steroid complex (primary response or early genes)
have been transcribed and translated, some of the proteins may
act to regulate the expression of other genes (secondary
response genes), etc.
So … you may have a series of different transcriptional events
over a time course with early, middle, and late events
A.
B.
C.
D.
E.
Amino Acid and Protein Structure
Formation of Aminoacyl tRNAs
Ribosome structure
Stages of Translation
Relationship between DNA, mRNA, and Protein Sequences
1.
Amino acid structure

Four different groups are attached to the central carbon atom (α-carbon)
 Hydrogen atom
 Amino group (-N+H3)
 Carboxylic acid group (-COO-)
 Side chain group (“-R”): 20 different amino acids, each with a different side
chain, are encoded by codons on mRNA
2.
Peptides and Proteins

Peptides are formed when a covalent peptide bond (an amide bond) is
formed between the carboxylic acid group of one amino acid and the
amino group of another amino acid.
2.
…
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Proteins are long peptides, over 50 amino acids long and typically much
longer (in the low 100s), and typically associated with some biological
function
The peptide chain of a protein folds into a specific three-dimensional
shape necessary for the activity of the protein.
The folding of the protein, and the chemistry of the protein’s active site,
are dependent on the amino acid sequence of the protein.
1.
2.
3.
Amino acids are covalently attached to the 3’ end of the
appropriate tRNAs. This is called the acceptor end.
The anticodon is a 3-base sequence on the anticodon loop of the
tRNA. It is complementary to the sequence of the codon on the
mRNA. The 5’ position is referred to as the “wobble base,”
meaning that it may pair up with more that one partner.
The reaction is catalyzed by an aminoacyl tRNA synthase. Each
tRNA has its own specific synthase enzyme.
4.
This is the reaction:
Amino acid + ATP + tRNA 
aminoacyl tRNA + AMP + PPi
5.
tRNA has a distinctive 3-D structure, described as a “cloverleaf,”
with hairpin loops and nonstandard bases
http://en.wikipedia.org/wiki/Transfer_RNA
1.
Prokaryotic ribosomes
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2.
Large subunit: 50 S
Small subunit: 30 S
Total size: 70 S
Eukaryotic ribosomes
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Large subunit: 60 S
Small subunit: 40 S
Total size: 80 S
http://en.wikipedia.org/wiki/Ribosome
1.
Initiation
a.
b.
c.
Requires the aid of initiation factor proteins
The small ribosome subunit binds to the 5’ end of mRNA. The proper
orientation is believed to be established by a sequence in the leader
region called the Shine-Dalgarno sequence (in prokaryotes) or similar
sequences.
An initiation codon (AUG) is oriented on the small ribosome subunit.
AUG is the codon for the amino acid methionine. Please note: Sequences
on mRNA are listed, by convention, in the 5’  3’ direction
…
1.
d.
e.
f.
A molecule of methionyl tRNA (met-tRNA) binds to the initiation codon
through codon-anticodon base pairing. This step requires GTP as an energy
source.
The large subunit binds to the small subunit to complete the initiation complex.
All initiation factors are released.
Some interesting facts:
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In prokaryotes, the methionine on the initiating methionyl tRNA is formylated (fmet-tRNA). In eukaryotes, it is not.
Not every AUG codon can be an initiation codon. Sequences in the mRNA leader
seem to indicate which AUG codons are initiation codons.
The initial methionine may be removed after translation (posttranslational
modification), so not every protein begins with a methionine.
2.
Elongation
a.
b.
c.
The ribosome/mRNA complex has two sites: the P site (to which the
growing peptide chain is attached) and the A site (where the next
aminoacyl tRNA binds). At the beginning of elongation, the met-tRNA
occupies the P site. The A site is ready to receive the next aminoacyl
tRNA.
The next aminoacyl tRNA binds to the ribosome/mRNA complex at the
A site.
An enzyme activity in the ribosome, peptidyl transferase, forms a peptide
bond between the carboxyl end of the growing peptide (on the P site) and
the amino end of the next amino acid (on the A site).
…
2.
d.
e.
f.
The tRNA on the P site, no longer attached to an amino acid, is released.
Another enzyme activity in the ribosome, called translocase, moves the
ribosome so that the peptidyl tRNA is transferred from the A site to the P
site. This process requires a GTP molecule as an energy source.
Now the A site is ready to accept the next aminoacyl tRNA.
3.
Termination
a.
b.
When the ribosome encounters a termination codon on the mRNA (UAA,
UAG, or UGA), elongation ceases.
Termination factors cause the ribosome, tRNA, and mRNA to dissociate
from the nascent protein chain.
Sequence
Nontemplate DNA strand:
5’ ATG TTT GCT AAG GAC ATC TAA 3’
Template DNA strand:
3’ TAC AAA CGA TTC CTG TAG ATT 5’
mRNA sequence:
5’ AUG UUU GCU AAG GAC AUC UAA 3’
Amino Acid Sequence:
(Amino end)
Met Phe Ala Lys Asp Ile
(Carboxyl end)
Be certain that you can read the genetic code table.
Be certain that you can read the genetic code table.