Download INTRODUCTION

Central dogma of Genetics
• A Gene is a DNA sequence
• A Gene encodes for a protein
• A protein could function as an enzyme or a biocatalyst
that is responsible for allowing chemical reactions to take
place in a cell.
• Many chemical reactions together produce a trait or
characteristic like flower color.
Genes to Traits
• Genes ----->proteins -----> Traits (Red flower color)
The Information Within the DNA Is Accessed
During the Process of Gene Expression
• Gene expression occurs in two steps
– Transcription
• The genetic information in DNA is copied into a nucleotide
sequence of ribonucleic acid (RNA)
– Translation
• The nucleotide sequence in RNA is converted (using the
genetic code) into the amino acid sequence of a protein
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TRANSCRIPTION
• Transcription literally means the act or process of
making a copy
• In genetics, the term refer to the copying of a DNA
sequence into an RNA sequence
• The structure of DNA is not altered as a result of
this process
– It can continue to store information
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Gene Expression

Structural genes encode the amino acids of a
polypeptide
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Transcription of a structural gene produces messenger
RNA, usually called mRNA
Polypeptide synthesis is called translation
The mRNA sequence determines the amino acids in the
polypeptide
The function of the protein determines traits
This path from gene to trait is called the central
dogma of genetics
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The central dogma of genetics
Signals the end of
protein synthesis
Gene Expression Requires Base Sequences

The strand that is actually transcribed (used as the template) is termed
the template strand

The opposite strand is called the coding strand or the sense strand as
well as the nontemplate strand
 The base sequence is identical to the RNA transcript
 Except for the substitution of uracil in RNA for thymine in DNA

Transcription factors recognize the promoter and regulatory sequences
to control transcription

mRNA sequences such as the ribosomal-binding site and codons direct
translation
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The Stages of Transcription
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Transcription occurs in three stages
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Initiation
Elongation
Termination
These steps involve protein-DNA interactions

Proteins such as RNA polymerase interact with DNA
sequences
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Transcription
Most of the promoter region is
labeled with negative numbers
Bases preceding
this are numbered
in a negative
direction
There is no base
numbered 0
Bases to the right are
numbered in a
positive direction
The conventional numbering system of promoters
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Sequence elements that play
a key role in transcription
The promoter may span a
large region, but specific
short sequence elements are
particularly critical for
promoter recognition and
activity level
Sometimes termed the
Pribnow box, after its
discoverer
\The conventional numbering system of promoters
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For many bacterial
genes, there is a good
correlation between
the rate of RNA
transcription and the
degree of agreement
with the consensus
sequences
The most commonly
occurring bases
Examples of –35 and –10 sequences within a variety of
bacterial promoters
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Initiation of Bacterial Transcription

RNA polymerase is the enzyme that catalyzes the
synthesis of RNA
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In E. coli, the RNA polymerase holoenzyme is
composed of
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Core enzyme
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Sigma factor
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Five subunits = a2bb’
One subunit = s
These subunits play distinct functional roles
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Binding of
factor protein to DNA double helix
Amino acids
within the a
helices hydrogen
bond with bases
in the promoter
sequence
elements
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Similar to the
synthesis of DNA
via DNA
polymerase
rho utilization site
Rho protein is
a helicase
r-dependent termination
r-dependent termination
•
r-independent termination is facilitated by two sequences in the RNA
– 1. A uracil-rich sequence located at the 3’ end of the RNA
– 2. A stem-loop structure upstream of the Us
URNA-ADNA hydrogen
bonds are very weak
This type is also called
intrinsic
r-independent termination
Stabilizes
the RNA pol
pausing
Eukaryotic RNA Polymerases
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Nuclear DNA is transcribed by three different RNA
polymerases
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RNA pol I
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Transcribes all rRNA genes (except for the 5S rRNA)
RNA pol II
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Transcribes all structural genes
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Thus, synthesizes all mRNAs
Transcribes some snRNA genes
RNA pol III
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Transcribes all tRNA genes
And the 5S rRNA gene
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Eukaryotic RNA Polymerases
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All three are very similar structurally and are
composed of many subunits
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There is also a remarkable similarity between the
bacterial RNA pol and its eukaryotic counterparts
Structure of
RNA polymerase
© From Patrick Cramer, David A. Bushnell, Roger D. Kornberg. "Structural Basis of
Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution." Science, Vol.
© From Seth Darst, Bacterial RNA polymerase.
292:5523, 1863-1876, June 8, 2001.
Current Opinion in Structural Biology.
Reprinted with permission of the author.
(a) Structure of a bacterial
RNA polymerase
5′
Structure of a eukaryotic
RNA polymerase II (yeast)
3′
Transcribed DNA
(upstream)
Lid
Exit
Clamp
Rudder
5′
Entering DNA
(downstream)
3′
Wall
5′
Mg2+
Bridge
Jaw
Catalytic
site
Transcription
NTPs enter
through a pore
(b) Schematic structure of RNA polymerase
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Usually an
adenine
• The core promoter is relatively short
– It consists of the TATA box
• Important in determining the precise start point for transcription
• The core promoter by itself produces a low level of
transcription
– This is termed basal transcription
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Usually an
adenine
• The core promoter is relatively short
– It consists of the TATA box
• Important in determining the precise start point for transcription
• The core promoter by itself produces a low level of
transcription
– This is termed basal transcription
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• Regulatory elements affect the binding of RNA polymerase
to the promoter
– They are of two types
• Enhancers
– Stimulate transcription
• Silencers
– Inhibit transcription
– They vary widely in their locations but are often found in
the –50 to –100 region
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A closed
complex
RNA pol II can
now proceed to
the elongation
stage
Released after
the open
complex is
formed
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Transcription termination
RNA polymerase II
• RNA polymerase II transcribes a gene
past the polyA signal sequence.
• The RNA is cleaved just past the
polyA signal sequence. RNA
polymerase continues transcribing
the DNA.
Possible mechanisms for Pol II termination
RNA polymerase II transcribes a gene
past the polyA signal sequence.
5′
3′
PolyA signal sequence
The RNA is cleaved just past the
polyA signal sequence. RNA
polymerase continues transcribing
the DNA.
5′
3′
3′
3′
5′
3′
5′
3′
5′
3′
Exonuclease catches
Exonuclease
up to RNA polymerase II
and causes termination.
5′
3′
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3′
RNA MODIFICATION
• In eukaryotes, the genes are interrupted: the coding
sequences, called exons, are interrupted by
intervening sequences or introns
• Transcription produces the entire gene product
– Introns are later removed or excised
– Exons are connected together or spliced
• This phenomenon is termed RNA splicing
– It is a common genetic phenomenon in eukaryotes
– Occurs occasionally in bacteria as well
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RNA MODIFICATION
• Aside from splicing, RNA transcripts can be modified
in several ways
– For example
• Trimming of rRNA and tRNA transcripts
• 5’ Capping and 3’ polyA tailing of mRNA transcripts
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This processing occurs
in the nucleolus
Functional RNAs that are key
in ribosome structure
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Found to contain both RNA
and protein subunits
Therefore, it is a ribozyme
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Covalently
modified bases
Pre-mRNA Splicing
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The spliceosome is a large complex that splices
pre-mRNA
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It is composed of several subunits known as
snRNPs (pronounced “snurps”)
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Each snRNP contains small nuclear RNA and a set of
proteins
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
Intron RNA is defined by particular sequences within the
intron and at the intro-exon boundaries
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The consensus sequences for the splicing of mammalian
pre-mRNA are shown here
Sequences shown in bold are
highly conserved
Corresponds to the boxed
adenine
Serve as recognition sites for the
binding of the spliceosome
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Intron loops out and
exons brought closer
together
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Cleavage may be
catalyzed by RNA
molecules within U2
and U6
Intron will be degraded and
the snRNPs used again
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Capping of pre-mRNA
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RNA editing
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Change in the nucleotide sequence of an RNA
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Can involve addition or deletion of particular bases
Can also occur through conversion of a base
First discovered in trypanosomes
Now known to occur in many organisms
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RNA editing
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