Molecular Biology

Transcription

Introduction

Gene expression
The central dogma of molecular biology
Solid arrows: transfers that occur in all cell
Dashed arrows: Special transfers
RNA-directed RNA polymerase
RNA-directed DNA polymerase (reverse transcriptase)
Missing arrows are information transfers never occur:
protein specifying either DNA, RNA, or protein
In other words, proteins can only be the recipients of genetic information

The classical flow of genetic information, often

referred to as "The Central Dogma" is:

Transcription
The synthesis of an RNA molecule from DNA
A complex process involving one of the group of RNA polymerase enzymes
RNA(n residues) + NTP

RNA(n + 1 residues) + PPi

RNA synthesis proceeds in a stepwise manner in the direction, that is, the
incoming nucleotide is appended to the free 3-OH group of the growing
RNA chain

Action of RNA polymerases

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

Function of the transcription bubble

RNA polymerase unwinds the DNA double helix, it separates a short segmet
(10 bp) to form transcription bubble, thereby permitting this portion to transiently
form a short DNARNA hybrid helix.
In bacteria, one species of this enzyme synthesizes all of the cells RNA.
Eukaryotic cells contain five different types of RNA polymerases that each
synthesize a different class of RNA.

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

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

Certain DNA Sequences.
Bacterial Promoters Are Relatively Simple
TATA box (Pribnow-Schaller box )

 Specific proteins known in prokaryotes as activators and repressors and in

eukaryotes as transcription factors bind to these control sites (promoter)
Transcriptional Termination Is a Relatively Simple Process

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.

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.

Transcription Factors

Two Models Can Explain the Assembly of the

Preinitiation Complex

A. Stepwise
TFIID, TFIIA, TFIIB, TFIIE, TFIIH, TFIIF, Med

B. Holoenzyme
Binding of a single protein complex:
pol II, Med and six GTFs

 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

The classical flow of genetic information, often

referred to as "The Central Dogma" is: