1.transcription 2012

The Central Dogma
The Flow of Information: DNA RNA

protein
DNA Replication
Transcription
Translation
Figure 12.2
Genes are made of DNA (Genotype).
They are expressed to make polypeptides
that determine the behaviour of the
organism (Phenotype)
The central dogma of molecular biology. Solid arrows indicate the types of
genetic information transfers that occur in all cells. Special transfers are indicated by
the dashed arrows: RNA-directed RNA polymerase occurs both in certain RNA
viruses and in some plants (where it is of unknown function); RNA-directed DNA
polymerase (reverse transcriptase) occurs in other RNA viruses; and DNA directly
specifying a protein is unknown but does not seem beyond the realm of possibility.
However, the missing arrows are information transfers the central dogma postulates
never occur: protein specifying either DNA, RNA, or protein. In other
words, proteins can only be the recipients of genetic information.
Gene expression. One strand of DNA directs the synthesis of RNA, a process
known as transcription. The base sequence of the transcribed RNA is complementary
to that of the DNA strand. The RNAs known as messenger RNAs (mRNAs) are
translated when molecules of transfer RNA (tRNA) align with the mRNA via
complementary base pairing between 3-nucleotide segments known as codons.
Each type of tRNA carries a specific amino acid. These amino acids are covalently
joined by the ribosome to form a polypeptide. Thus, the sequence of bases in DNA
specifies the sequence of amino acids in a protein.
The synthesis of an RNA molecule from DNA is a complex process involving one of
the group of RNA polymerase enzymes and a number of associated proteins.
The general steps required to synthesize the primary transcript are:
initiation,
elongation, and
termination.
Most is known about initiation. A number of DNA regions (generally located upstream
from the initiation site) and protein factors that bind to these sequences to regulate the
initiation of transcription have been identified.
Certain RNAsmRNAs, in particularhave very different life spans in a cell. It is
important to understand the basic principles of messenger RNA synthesis
and metabolism, for modulation of this process results in altered rates of protein
synthesis and thus a variety of both metabolic and phenotypic changes.
This is how all organisms adapt to changes of environment. It is also how
differentiated cell structures and functions are established and maintained.
The RNA molecules synthesized in mammalian cells are made as precursor
molecules that have to be processed into mature, active RNA. Errors or changes in
synthesis, processing, and splicing of mRNA transcripts are a cause of disease.
RNA Is Synthesized from a DNA Template

by an RNA Polymerase
The processes of DNA and RNA synthesis are similar in that they involve
(1) the general steps of initiation, elongation, and termination with 5' to 3' polarity;
(2) large, multicomponent initiation complexes; and
(3) adherence to Watson-Crick base-pairing rules.
These processes differ in several important ways, including the following:
(1) ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides;
(2) U replaces T as the complementary base pair for A in RNA;
(3) a primer is not involved in RNA synthesis;
(4) only a portion of the genome is transcribed or copied into RNA, whereas the
entire genome must be copied during DNA replication;
(5) there is no proofreading function during RNA transcription.
Watson and Crick model of the double-helical structure of the B form of DNA.
Messenger RNA
The relationship between the sequences of an RNA transcript and its gene, in which
the coding and template strands are shown with their polarities.
The RNA transcript with a 5' to 3' polarity is complementary to the template strand
with its 3 to 5' polarity. Note that the sequence in the RNA transcript and its polarity
is the same as that in the coding strand, except that the U of the transcript replaces
the T of the gene.
The Template Strand of DNA Is Transcribed

The strand that is transcribed or copied into an RNA molecule is referred to as the
template strand of the DNA. The other DNA strand, the non-template strand, is
frequently referred to as the coding strand of that gene. It is called this because,
with the exception of T for U changes, it corresponds exactly to the sequence of the
RNA primary transcript, which encodes the (protein) product of the gene.
In the case of a double-stranded DNA molecule containing many genes, the template
strand for each gene will not necessarily be the same strand of the DNA double helix
Thus, a given strand of a double-stranded DNA molecule will serve as the template
strand for some genes and the coding strand of other genes.
Note that the template strand is always read in the 3' to 5' direction.
The Prokaryotic Transcription

DNA-Dependent RNA Polymerase Initiates Transcription at a Distinct Site, the
Promoter
E coli RNAP consists of a core complex of : 2, often termed E, associates

with a spesific protein factor (sigma factor) to form holoenzyme, 2 or E
The transcription "bubble" is an approximately 20-bp area of melted DNA, and the
entire complex covers 3075 bp, depending on the conformation of RNAP.
The transcription cycle in bacteria
Bacterial RNA transcription is described in five steps:

(1) Template binding: RNAP binds to DNA and locates a promoter (P) melts
the two DNA strands to form a preinitiation complex (PIC).
(2) Chain initiation: RNAP holoenzyme (core + one of multiple factors)
catalyzes the coupling of the first base (usually ATP or GTP) to a second
ribonucleoside triphosphate to form a dinucleotide.
(3) Promoter clearance: RNAP undergoes a conformational change after
RNA
chain length reaches 1020 nt and then is able to move away from the
promoter, transcribing down the transcription unit.
(4) Chain elongation: Successive residues are added to the 3'-OH terminus of
the nascent RNA molecule.
(5) Chain termination and release: The completed RNA chain and RNAP are
released from the template. The RNAP holoenzyme re-forms, finds a
promoter, and the cycle is repeated.
Bacterial promoters, E Coli
(Pribnow box)
The Fidelity & Frequency of Transcription Is Controlled by Proteins Bound to

Certain DNA Sequences.
Bacterial Promoters Are Relatively Simple
Rho-dependent transcription termination signals
Bacterial transcription termination signal contains: inverted repeat ( dyad symetry)

(the two boxed areas) followed by a stretch of AT base pairs.
The inverted repeat, when transcribed into RNA, can generate the hairpin secondary
structure in the RNA transcript. Formation of this RNA hairpin causes RNAP to pause
and subsequently the termination factor interacts with the paused polymerase and
somehow induces chain termination.
Transcription continues into the AT region, and with the aid of the termination
protein the RNA polymerase stops, dissociates from the DNA template, and releases
the nascent transcript.
Eukaryotic Promoters Are More Complex
Schematic diagram showing the transcription control regions in a hypothetical

mRNA-producing, eukaryotic gene transcribed by RNA polymerase II. Such a gene
can be divided into its coding and regulatory regions, as defined by the transcription
start site (arrow; +1).
Proximal and distal cis elements are bound by trans -acting transcription factors, in
this example: Sp1 and CTF (also called C/EBP, NF1, NFY). These cis elements can
function independently of orientation (arrows).
Basal Transcription Complex

Formation of the basal transcription complex begins when TFIID binds to the TATA
box. It directs the assembly of several other components by protein-DNA and
protein-protein interactions; TFIIA, B, E,F, H, and polymerase II (pol II).
The entire complex spans DNA from position 30 to +30 relative to the initiation site
(+1, marked by bent arrow)
Promoter Accessibility
and Hence PIC
Formation
Is Often Modulated
by Nucleosomes
Nucleosome eviction by
chromatin-active coregulators
facilitates PIC formation and
transcription.
Specific Signals Regulate Transcription Termination

The signals for the termination of transcription by eukaryotic RNA polymerase II are
only poorly understood.
Less is known about the termination process or whether specific termination
factors similar to the bacterial factor are involved.
In eukaryotes, the mechanisms for terminating transcription appear to differ for
each of the three RNA polymerases:
- Transcription of pre-rRNA genes by RNA polymerase I is terminated by a
mechanism that requires a polymerase-specific termination factor. This DNAbinding protein binds downstream of the transcription unit, unlike the E. coli Rho
factor, which is a RNA-binding termination factor.
- RNA polymerase III terminates after polymerizing a series of U residue,
and does not require an stem-loop secondary structure in the RNA transcript.
- RNA polymerase II can terminate at multiple sites located over a distance
of 0.52 kb beyond the poly(A) addition site and termination is coupled to the
process that cleaves and polyadenylates the 3 end of a transcript, associates with
the phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II.
Transcription Factors
Two Models Can Explain the Assembly of the

Preinitiation Complex
(recruitment hypothesis)
A. Stepwise
TFIID, TFIIA, TFIIB, TFIIE, TFIIH, TFIIF, Med
B. Holoenzyme
Binding of a single protein complex:
pol II, Med and six GTFs
In vitro assembly of RNA

polymerase II
preinitiation complex.
Posttranscriptional modification of mRNA

RNA MOLECULES ARE USUALLY PROCESSED BEFORE THEY BECOME
FUNCTIONAL
The Coding Portions (Exons) of Most Eukaryotic Genes Are Interrupted
by Introns
Introns Are Removed & Exons Are Spliced Together
Alternative Splicing Provides for Different mRNA
Alternative Promoter Utilization Provides a Form of Regulation
Messenger RNA (mRNA) Is Modified at the 5' & 3' Ends
Regulation of Eukaryotic Transcription

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The Central Dogma

The Flow of Information: DNA RNA

RNA Is Synthesized from a DNA Template

The Template Strand of DNA Is Transcribed

The Prokaryotic Transcription

E coli RNAP consists of a core complex of : 2, often termed E, associates

The transcription cycle in bacteria

Bacterial RNA transcription is described in five steps:

Bacterial promoters, E Coli

The Fidelity & Frequency of Transcription Is Controlled by Proteins Bound to

Rho-dependent transcription termination signals

Bacterial transcription termination signal contains: inverted repeat ( dyad symetry)

Eukaryotic Promoters Are More Complex

Schematic diagram showing the transcription control regions in a hypothetical

Basal Transcription Complex

Specific Signals Regulate Transcription Termination

Two Models Can Explain the Assembly of the

In vitro assembly of RNA

Posttranscriptional modification of mRNA

Regulation of Eukaryotic Transcription

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