DEFINITION: TRANSCRIPTION IS THE

UNIT 5 SYNTHESIS OF A RNA MOLECULE FROM A

DNA TEMPLATE

The coding strand is the

“sense” strand which
contains the gene

• RNA IS TRANSCRIBED FROM ONE DNA STRAND

• DIFFERENT GENES MAY BE TRANSCRIBED FROM ONE OR
THE OTHER OF THE TWO STRANDS
TRANSCRIPTION LAYOUT

• Structure and clases of RNA

• Structure of DNA template (transcription unit / gene) in

bacteria and eukaryotes.

• The basal transcription apparatus in prokaryotes and

eukaryotes: RNA polymerases and accesory proteins.

• Phases of transcription in prokariotes and eukariotes:

Initiation, Elongation, and Termination

• Posttranscriptional mRNA modifications

• Summary Under the electron microscope DNA

molecules undergoing trasncription exhibit
“Christmas-like tree structures”.
 CLASSES OF RNA MOLECULES

RNA STRUCTURE: RNA differs from DNA in that RNA possesses a hydroxyl group on
the 2´-carbon atom of its sugar, contains U instead of T and is normaly single strand

Class of RNA (**) Cell Type Location Function

Ribosomal RNA (rRNA) Bacterial & Eukaryotic Cytoplasm Ribosome structure and function

Messenger RNA (mRNA) Bacterial & Eukaryotic Nucleus & Cytoplasm Carries of genetic code for proteins

Transfer RNA (tRNA) Bacterial & Eukaryotic Cytoplasm Incorporates AA into polypeptide chain
MicroRNA* (miRNA) Eukaryotic Cytoplasm Inhibits translation of mRNA

Small Interfering RNA*(siRNA) Eukaryotic Cytoplasm Degradation of mRNA

(*) RNA of interference

(**) Other RNA in Eukaryotes: small nuclear RNA (RNA splicing); small nucleolar RNA (processing and assembly of tRNA).
STRUCTURE OF THE BACTERIAL (TRANSCRIPTION UNIT) GENE

• Transcription unit: strecht of DNA that

encodes an RNA molecule (RNA-coding
region and terminator) and the sequence
necessary for its transcription (promoter).

• Promoter: DNA sequence adjacent to a

gene and required for transcription. It
contains “consensus sequences”.

• “Consensus sequences" are short stretches

of nucleotides common to most promoters.
Spacing and location relative to the start site
are similar in most promoters.

Notice: When DNA sequences are written out,

often the secuence of only one of the two strands
is listed (usually 5´→3´ as the transcribed RNA).
BASAL TRANSCRIPTION APPARATUS IN BACTERIA

• Bacterial cells possess a single RNA

polymerase that catalyzes all clases of
bacterial RNAs.

• The sigma factor allows bacterial RNA

polymerase to bind to the “consensus
sequences” of the promoter.

• The sigma factor positions the RNA Pol

at the correct start site.
 TRANSCRIPTION IN BACTERIA
Initiation: location of consensus sequences
positions RNA polymerase over the correct start
site for RNA sysnthesis.

Elongation: Polymerase begin to transcribing

downstream. Transcription is 5´→3´; each
nucleotide is joined to the 3´-OH group of the last
nucleotide added to the growing RNA molecule.

Termination: Transcription ends after RNA pol

transcribes a terminator sequence (an inverted
repeat followed by 6x Adenines).
THE “TERMINATOR” HAIRPIN LOOP

Intrinsic termination

When RNA polymerase encountes a termination

sequence it pauses, and Rho catchs up.
The helicase Rho unwinds the DNA-RNA pairing
 EUKARYOTIC RNA POLYMERASES

The eukaryotic nucleus contains three major classes of DNA-dependent

RNA polymerase. Enzymes of the different classes have different
transcriptional specificity, and recognize different types of promoters.
REGULATION OF THE “GIANT” RNA POLYMERASE II
• The basal transcription apparatus
consists of the RNA pol II, “general”
transcription factors (TFII A to H) and
protein mediators (TBP).

• Recognition sites include the core

promoter, the proximal (regulatory)
promoter and the enhancer.

Note that there are both “general” and “specific” transcription factors
 INTITIATION OF TRANSCRIPTION IN EUKARYOTES

• Sigma function is replaced by general

transcription factors (TF) and mediators (TBP).

• The core promoter is the DNA sequence

recognized by the RNA polymerase as a correct
start site for the synthesis of RNA.

• The regulatory promoter (adjacent to the core

promoter) and enhancer regulate when and
what level a gene is expressed.

• The enhancer can be located hundreds, or even

several thousand base pairs from the promoter
and still bind with activators to increase the
amount of RNA synthesized from a promoter.

Notice that the RNA polymerase II does not directly interact with the promoter as it is a “giant” protein complex
 REGULATORY ELEMENTS OF EUKARYOTIC GENES CONTAIN CAAT AND GC BOXES
A regulatory sequence is a segment of a nucleic acid molecule which is
capable of increasing or decreasing the expression of specific genes

The CAAT box contains the sequence CAAT or CCAAT and the GC box has the sequence GGGCGG.

Note that different types of regulatory elements require different proteins that recognizes the consensus sequences (boxes)
 Termination: Cleveage of mRNA just past a polyA signal
ELONGATION AND TERMINATION sequence produces a 5´end to which an exonuclease can attach.
IN EUKARYOTIC CELLS Exonuclease degrades RNA molecule in the 5´→3´direction.

Elongation: The DNA double helix enters de RNA Pol II

through a groove and unwinds. The DNA-RNA duplex is bent
at a right angle, which positions the 3´end at the active site.
New NTPs are added to the 3´end of RNA.
 POSTRANSCRIPTIONAL MODIFICATIONS TO EUKARYOTIC “PRE-mRNA”
Modification Function
Addition of 5´cap Increases mRNA stability and facilitates ribosome binding
3´cleavage and addition of poly(A) tail Increases mRNA stability and facilitates ribosome binding
RNA splicing Remove introns and allows for multiple proteins to be produce by alternative splicing
RNA editing Alters nucleotide mRNA sequence

Poly(A) tail

5´cap
RNA PROCESSING: THE SPLICING OF Pre-mRNA MOLECULES
Introns: noncoding sections of an RNA
transcript, that are spliced out before the
RNA molecule is translated into a protein.
Introns interrupts exons.

RNA splicing occurs in the nucleus before

mRNA is moved to cytoplasm. Spliceosomes
consists of small nuclear RNA and proteins.

Mature mRNA regions: 5´untranslated region

(5´-UTR), protein coding region, and
3´untranslated region (3´-UTR).
ALTERNATIVE pre-mRNA SPLICING MULTIPLE 3´CLEAVAGE SITES

CGRP: Calcitonin-gene-related peptide

• Alternative splicing enables exons to be spliced

together in different combinations to yield mRNAs.

• Multiple 3´cleavage sites allow pre-mRNA to be cleaved

at different sites.

• Both results in different proteins from the same gene

and provides opportunity for gene regulation.
mRNA TRANSCRIPTION IN EUKARYOTIC CELLS IS MORE COMPLEX THAN IN BACTERIA
Remember that:
In contrast to Bacteria, Eukaryotes possess three classes of RNA pol

The eukaryotic RNA Pol II protein has more complexity

Posttranscriptional processing of mRNA occurs only in eukaryotes

• Bacterial RNA Pol directly binds to a promoter

region and transcription unit synthetizes
mRNA at high rates.

• Basic transcription unit in eukaryotes requires

accesory proteins to initiate transcription at
low rates. Tissue-specific transcription
factors enhance mRNA synthesis.

• Termination is mediated by hairpin loops in

bacteria and by polyA sequences in
Eukaryotes