Chapter 13

Transcription, RNA and RNA Processing

Translation: Protein synthesis; process by which the amino acid sequence of a polypeptide is
synthesized on a ribosome according to the sequence of an mRNA

Transcription: The process by which the information contained in a template strand of DNA is
copied into a ssRNA molecule of complementary base sequence

Transcription process
1.The process by which mRNA is made from a DNA template (or “antisense”)

2.Carried out by RNA polymerase in a 5’-3’ direction (RNA) Polymerase moves along DNA in 3'
—> 5' direction (toward 5' end)

3.Only 1 strand of DNA duplex serves as the template for a given gene

4.RNAn + NPPP —> RNAn+1 + PPi (—> 2 Pi)

5.RNA polymerases bind to sites on DNA called promoters

6. 2 short stretches of DNA that are similar from one gene to another (-35 region TTGACA
& -10 region TATAAT )

RNA polymerases do not require the presence of a primer

The resulting mRNA molecule has the same sequence as the non-template (or “sense”) strand,
except for replacing thymine with uracil.
Only one DNA strand is transcribed for any one gene
Different genes can be transcribed on different strands though

Arrangement of promoters (green) and termination sites (orange) in a segment of DNA

Messenger RNA (mRNA) encodes the amino acid sequence of a polypeptide. mRNAs are the
transcripts of protein-coding genes

Transfer RNA (tRNA) brings amino acids to the ribosomes during translation

Ribosomal RNA (rRNA) complexes with ribosomal proteins to make the ribosome, the organelle
that translates the mRNA

Small nuclear RNA (snRNA) complexes with snRNPs, involved in eukaryotic splicing

3 stages of transcription
1.Chain initiation
RNA polymerase binds to initiate transcription
 Initiation - binding of RNA polymerase to double-stranded DNA. RNA polymerase binds
at a sequence of DNA called the promoter

2.Chain elongation
Only the template strand is transcribed
The transcription bubble is ~15 nt long with 8-9 nt paired with the 3' end of the RNA
 Elongation - the covalent addition of nucleotides to the 3' end of the growing
polynucleotide chain

3.Termination
RNA polymerase reaches a terminator sequence and the RNA and polymerase are released
 Termination - the recognition of the transcription termination sequence and the release
of RNA polymerase
 Prokaryotic Gene Structure
Gene: Transcriptional Unit

+1 Open Reading Frame (ORF)

Promoter RNA coding Sequence Terminator
5’ 3’
3’ 5’

RNA transcript
Template strand

5’ UTR 3’ UTR

Prokaryotic RNA Polymerase

RNA Polymerase Holoenzyme

Prokaryotic Promoter (1)

E. coli promoters have conserved (2) sequences as -10 and -35 nt

The -35 and -10 regions determine promoter strength.

Nucleotide sequences in promoter regions of several E. coli genes

Prokaryotic Promoter (3)

Initiation

Elongation

Termination

Self-termination sequences contain a hairpin and a U-rich region

Some terminators also require a terminator protein

Nucleotide sequence of the transcription–termination region for the set of tryptophan-

synthesizing genes in E. coli
The 3' terminus of the RNA transcript, folded to form a stem-and-loop structure

Self-Termination

Termination Stem-Loop
Termination

Rho-dependent:
Absence of poly U sequence
Has a G-C rich region upstream of termination, may or may not be palindromic
Hexamer of 6 identical units
Rho binds to rut (rho utilization) site in 3’ UTR
Travels along mRNA at same speed as RNA polymerase
When polymerase pauses at the G-C rich region, rho catches up to it and unwinds DNA-RNA
hybrid causing polymerase to fall off
Eukaryotic Transcription
Similar but more complicated process than in prokaryotes

4 RNA polymerases

RNAP II transcribes mRNA for protein encoding genes

Takes place in the nucleus

Pre-mRNA molecule that is modified and processed to produce a functional mRNA molecule

Prokaryotes have one type RNAP that carry out all transcriptional reactions
Eukaryotic organisms have 4 types of RNAPs
RNAP I: transcribes most of the rRNAs genes
RNAP II: transcribes the structural genes (most important)
RNAP III: transcribes the tRNAs genes, 5S rRNA genes and snRNA genes
RNAP IV: transcribes some siRNA’s in plants

Mitochondrial RNAP: Transcribes genes located on the mitochondrial chromosome

Eukaryotic cells makes a host of other RNAs

Small cytoplasmic RNAs (scRNAs)

Small nucleolar RNAs (snoRNAs)

Small interfering RNAs (siRNAs)

Micro RNAs (miRNAs)

Piwi-interacting RNA (Pi-RNA)

(found in mammalian testes. Similar to miRNA & siRNA)
 Initiation
• RNAP binds to promoter region
• Requires accessory proteins to help RNAP II to
recognize and bind promoter sequence
• Basal promoter elements:
– TATA box at position -25, TATAAA consensus
– Initiator element (Inr), PyPyAN(T/A)PyPy consensus
sequence
– CAAT box at position -75
– GC box at position -90
(GGGCGG consensus) Proximal Promoter
elements
 RNA polymerase II promoters contain a TATA
box to signal +1

Some human TATA-box genes showing sequences in the core

promoter region near the transcription start site

• General TFs - bind at core promoter sites in association with RNA polymerase

• Sequence-specific TFs - bind to various regulatory sites of particular genes;

– they either stimulate (transcriptional activators) transcription of adjacent genes

or

– inhibit (transcriptional repressors) transcription of adjacent genes

Basal Transcription Factors

 TFIID, binds to TATA box

– Comprised of TBP (TATA binding protein) and TAFs (TBP associated factors)

– Forms pre-initiation complex

 Binding of TFIIB and recruitment of RNAP II and TFIIF

– Minimal transcription initiation complex

 TFIIE and TFIIH assemble to forming the complete initiation complex

 Basal level of transcription
Transcription Factor Structure
The presence of domains in TF's
 One domain (the DNA-binding domain) recognizes & binds to specific DNA base pair
sequence

 Other domain (activation domain) regulates transcription by interacting with other

proteins

 A major distinction between transcription in prokaryotes & eukaryotes is the

requirement in eukaryotes of transcription factors (TFs)

 (TFs) play a role in virtually every aspect of transcription process from binding
polymerase to DNA template, initiation of transcription, elongation &termination
Enhancer Elements

Enhancers are regulatory elements that when bound by Activators, a type of transcription
factor, result in increased transcriptional activity

Enhancers can be upstream, downstream or within the gene, can function in either orientation
and can be VERY far away

Transcription Factor Motifs

 Most motifs contain a segment (often an a-helix) that is inserted into the major groove
of DNA, where it recognizes the sequence of base pairs that line the groove

 Protein binding to DNA is achieved by a specific combination of van der Waals forces,
ionic bonds & H bonds between amino acid residues & various parts of DNA, including
backbone

 Distal promoter elements to distinguish them from the proximal elements situated
closer to the gene

 Enhancers even more distant DNA elements (than proximal or distal promoter elements)

• Coactivators that interact with the basal transcription machinery – transcription is

accomplished through recruitment & subsequent collaboration of large protein complexes

• Coactivators that alter chromatin structure – eukaryotic DNA is organized into

nucleosomes; how can nonhistone proteins (TFs, RNA polymerases) interact with DNA
tightly associated with core histones?
Silencers
 Similar to Enhancers except when bound by transcription factors known as Repressors,
they decrease the level of transcription

 Variations in the level of activators and repressors in different cell types give tissue
specific expression of genes

Prokaryotic vs Eukaryotic
Prokaryotes have Polycistronic mRNAs

 Prokaryotes have Coupled RNA transcription and protein synthesis

Cytoplasmic membrane does not separate the events of transcription and translation
 Eukaryote mRNAs have Introns
 RNAP types
Transcription Key Terms
7-methylguanosine cap
A structure consisting of GTP and methylated sugars that is added to the 5′ ends of
eukaryotic mRNAs.
alternative splicing
The generation of different mRNAs by varying the pattern of pre-mRNA splicing.
chromatin immunoprecipitation
A method for determining regions of DNA that bind transcription factors within a cell.
chromatin remodeling factor
A protein that disrupts chromatin structure by altering the contacts between DNA and
histones.
cis-acting control element
A regulatory DNA sequence that serves as a protein binding site and controls the
transcription of adjacent genes.
coactivator
A protein that interacts with a transcription factor to stimulate transcription.
corepressor
A protein that associates with repressors to inhibit gene expression, often by modifying
chromatin structure.
CTCF
An architectural protein involved in regulating the organization of chromatin.
DNA-affinity chromatography
A method used to isolate DNA-binding proteins based on their binding to specific DNA
sequences.
DNase hypersensitive site
A nucleosome-free region of chromatin, characteristic of promoters and enhancers,
that is sensitive to digestion by DNase.
electrophoretic-mobility shift assay
An assay for the binding of a protein to a specific DNA sequence.
enhancer
A transcriptional regulatory sequence that can be located at a site distant from the
promoter.
epigenetic inheritance
The transmission of information from parent to progeny that is not contained within
the sequence of DNA.
general transcription factor
A transcription factor that is part of the general transcription machinery.
genomic imprinting
The regulation of genes whose expression depends on whether they are maternally or
paternally inherited, controlled by DNA methylation.
helix-loop-helix
A transcription factor DNA-binding domain formed by the dimerization of two
polypeptide chains. The dimerization domains of these proteins consist of two helical
regions separated by a loop.
helix-turn-helix
A transcription factor DNA-binding domain in which three or four helical regions
contact DNA.
histone acetylation
The modification of histones by the addition of acetyl groups to specific lysine residues.
homeodomain
A type of DNA binding domain found in transcription factors that regulates gene
expression during embryonic development.
insulator
A sequence that divides chromatin into independent domains and prevents an
enhancer from acting on a promoter in a separate domain.
leucine zipper
A protein dimerization domain containing repeated leucine residues that is found in
many transcription factors.
long noncoding RNA (lncRNA)
An RNA longer than 200 nucleotides that regulates gene expression.
Mediator
A complex of proteins that stimulates transcription of eukaryotic protein-coding genes
and allows them to respond to gene-specific regulatory factors.
mRNP
Messenger ribonucleoprotein particles, consisting of mRNA associated with proteins.
nonsense-mediated mRNA decay
Degradation of mRNAs that lack complete open-reading frames.
operator
A regulatory sequence of DNA that controls transcription of an operon.
operon
A group of adjacent genes transcribed as a single mRNA.
poly-A tail
A tract of about 200 adenine nucleotides added to the 3′ ends of eukaryotic mRNAs.
polyadenylation
The process of adding a poly-A tail to a pre-mRNA.
Polycomb proteins
A complex of repressor proteins that methylate histone H3 lysine 27.
pre-mRNA
The primary transcript, which is processed to form messenger RNAs in eukaryotic cells.

pre-rRNA
The primary transcript, which is cleaved to form individual ribosomal RNAs (the 28S,
18S, and 5.8S rRNAs of eukaryotic cells).
pre-tRNA
 The primary transcript, which is cleaved to form transfer RNAs.
promoter
A DNA sequence at which RNA polymerase binds to initiate transcription.
repressor
A regulatory molecule that blocks transcription.
ribozyme
An RNA enzyme.
RNA editing
RNA processing events other than splicing that alter the protein coding sequences of
mRNAs.
RNA polymerase
An enzyme that catalyzes the synthesis of RNA.
RNase P
A ribozyme that cleaves the 5′ end of pre-tRNAs.
self-splicing
The ability of some RNAs to catalyze the removal of their own introns.
small nuclear ribonucleoprotein particle (snRNP)
Complex of an snRNA with proteins.
small nuclear RNA (snRNA)
A nuclear RNA ranging in size from 50 to 200 bases.
spliceosome
A large complex of snRNAs and proteins that catalyzes the splicing of pre-mRNAs.
steroid hormone receptor
A transcription factor that regulates gene expression in response to steroid hormones.
TATA box
A regulatory DNA sequence found in the promoters of many eukaryotic genes
transcribed by RNA polymerase II.
TATA-binding protein (TBP)
A basal transcription factor that binds directly to the TATA box.
TBP-associated factor (TAF)
A polypeptide associated with TBP in the general transcription factor TFIID.
transcription factor
A protein that regulates the activity of RNA polymerase.
transcriptional activator
A transcription factor that stimulates transcription.
zinc finger domain
A type of DNA binding domain consisting of loops containing cysteine and histidine
residues that bind zinc ions.