2. Gene Expression
Gene expression process by which
a genes information is converted
into the structures and functions of
a cell by a process of producing a
biologically functional molecule of
either protein or RNA (gene
product) is made.
Gene expression is assumed to be
controlled at various points in the
sequence leading to protein
3. Gene Expression
Every cell of the body (with a few exceptions)
contains a full set of chromosomes and identical
genes. Only a fraction of these genes is turned on,
however, and it is the subset that is “expressed”
that confers unique properties to each cell type.
The proper expression of a large number of genes
is a critical component of normal growth and
development and the maintenance of proper
Disruptions or changes in gene expression are
responsible for many diseases.
4. Protein synthesis is the process in which cells build protein from
information in DNAin two major steps:
Synthesis of an RNA that
is complementary to one
of the strands of DNA
according to instruction
stored along a specific
sequence (a gene) of a
Ribosomes read a
messenger RNA and make
protein according to its
The overall scheme is similar in bacteria and eukaryotes, but
there are significant difference; transcription initiation and
transcription termination especially with added complexity of the
eukaryotic transcription initiation system.
8. Overall architecture of RNAPs from bacteria (Thermus aquaticus (1HQM)
Minakhin et al., 2001), archaea (Sulfolobus shibatae (2Y0S) Wojtas et al.,
2011) and eukaryotes (Saccharomyces cerevisiae (1Y1V) Kettenberger et
Overall architecture of RNAPs from bacteria (Thermus
aquaticus (1HQM) Minakhin et al., 2001), archaea (Sulfolobus
shibatae (2Y0S) Wojtas et al., 2011) and eukaryotes (Saccharomyces
cerevisiae (1Y1V) Kettenberger et al., 2004) http://www.biologie.uni-
9. Transcription Enzymes
Eukaryotic nuclei contain three RNA polymerases. RNA
polymerase I is found in the nucleolus; RNA polymerase II &III
are located in the nucleoplasm. The three nuclear RNA
polymerase have different roles in transcription.
Polymerase I makes a large precursor to the major rRNA
(28S,18S and 5.8S rRNA in vertebrates).
Polymerase II synthesizes hnRNAs, which are precursors to
mRNAs. It also make most small nuclear RNAs (snRNAs).
Polymerase III makes the precursor to 5SrRNA, the tRNAs and
several other small cellular RNAs.
Prokaryotes have one type of RNA polymerase for all types of RNA.
10. The key player in the transcription process is RNA polymerase. The E-
coli enzyme is composed of a core, which contains the basic
transcription machinery, and a - factors which directs the core to
transcribe specific gene.
11. Subunit composition of the RNAPs from the
three domains of life (modified from doctoral thesis Zeller M. E.)
Subunit composition of the RNAPs
12. Transcription has three phases: initiation,
elongation, termination. The following is an
outline of the three step in bacteria…
13. Prokaryote genes are grouped in operons
Transcription is a vital control point in the expression of many
RNA polymerase directs transcription. RNA polymerase is the
signal that control transcription. RNA polymerase docks at
a promoter and continue as the polymerase elongates the
RNA chain and ends when the polymerase reaches a
terminator and release the finished transcript.
15. Prokaryotic Transcription Initiation
Represented as four steps:
Formation of a closed promoter complex.
Conversion of the closed promoter to an open promoter
Polymerizing the first few nucleotides (up o 10) while the
polymerase remain at the promoter.
16. The - factor allows initiation of transcription by causing the RNA
polymerase holoenzyme to bind tightly to a promoter. This tight binding
depends on local melting of the DNA to form an open promoter complex
and is stimulated by . The - factor can therefore select which genes will
Bacterial promoters contain two regions centered at -10 and –35 bp
upstream of the transcription start site. In E-coli, there is bear a greater or
lesser resemblance to two consensus sequences: TATAAT and TTGACA,
The RNA polymerase binding causes the unwinding of the DNA
double helix which expose at least 12 bases on the template.
This is followed by initiation of RNA synthesis at this starting point.
The transcription bubble moves with the polymerase, exposing the
template strand so it can be transcribed.
The RNA polymerase starts building the RNA chain, it assembles
ribonucleotides triphosphates: ATP; GTP; CTP and UTP into a strand
After the first nucleotide is in place, the polymerase joins a second
nucleotide to the first, forming the initial phosphodiester bond in the
After has participated in initiation, it appears to dissociate from the
core polymerase to carry out elongation. can be reused by different
The core continues to elongate the RNA, adding one nucleotide after
another to the growing RNA chain.
RNA polymerase directs the sequential binding of riboncleotides to the
growing RNA chain in the 5'-3' direction. RNA polymerase moves
along DNA template, and the bubble of melted DNA moves with it.
This transcription bubble is 10-18 bases long and contain an RNA-
DNA hybrid about 9bp long.
Each ribonucleotide is inserted into the growing RNA strand following
the rules of base pairing. This process is repeated till the desired RNA
length is synthesized……………………..
Some regions on the DNA that signal termination
(terminators) are recognized by RNA polymerase. Two kinds
Intrinsic terminators, function with the RNA polymerase
by itself without help of other proteins.
Rho dependent termination.
The result is that the RNA transcript dissociate from RNA
polymerase and DNA and so stop transcription.
22. The general transcription factors
combine with RNA polymerase
form a preinitiation complex that is
competent to initiate transcription
as soon as nucleotide are available.
23. Preinitiation Complex
First, an RNA polymerase along with general transcription
factors binds to the promoter region of the gene to form a
closed complex called the preinitiation complex.
Preinitiation complex contains:
Core Promoter Sequence
Activators and Repressors.
24. Transcription starts upstream from the first coding sequence at the
transcription initiation site.
RNA polymerase recognizes a promoter. Eukaryotic Promoter lies
upstream of the gene. There are several different types of promoter
found in human genome, with different structure and different regulatory
One important promoter sequence is the TATA box, a conserved region
rich in adenines and thymines, approximately 20-30 bp upstream of the
start site of transcription. The TATA box appears to be important for
determining the position of the start of transcription.
The assembly of the preinitiation complex on each kind of eukaryotic
promoter e.g. (class II promoters recognized by RNA polymerase II)
begins with the binding of an assembly factor to the promoter. With
TATA containing class II and presumably class III) promoters, this factor
is TBP, but other promoters have their own assembly factors.
This tight binding involves the formation of an open promoter
complexes in which the DNA at the transcription start site has melted to
allow the polymerase to read it.
Eukaryotic Transcription Initiation
25. Source: www.ucl.ac.uk
26. TranscriptionFactors for RNApolymerase II (human cells)
mass (kDa) Functions
TFIID: TBP 1 38 Recognize core promoter (TATA) TFIIB
TFIID: TAFs 12 15-250 Recognize core promoter (non-TATA);
Positive and negative regulation
RNA Pol II?
TFIIA 2 12, 19, 35 Stabilize TBP-DNA binding; Anti-repression
TFIIB 1 35 Select start site for RNA Pol II RNA PolII-TFIIF
RNA Pol II 12 10-220 Catalyze RNA synthesis TFIIE
TFIIF 2 30, 74 Target RNA PolII to promoter; destabilize
non-specific interactions between PolII and
TFIIE 2 34, 57 Modulate TFIIH helicase, ATPase and kinase
activities; Directly enhance promoter
TFIIH 9 35-89 Helicase to melt promoter; CTD kinase;
Source: Roeder, R.G. (1996) TIBS 21: 327-335
27. Transcription Factors
The class II preinitiation
polymerase II and six
transcription factors TFIIA,
TFIIB, TFIID, TFIIE, TFIIF,
TFIIH. The class general
transcription factors and
RNA polymerase bind in
specific order to the growing
preintiation complex (at
least in vitro).
28. Mediators,; another collection of proteins and can be considered as a general transcription factor, because it
is a part of most, if not all class II preintiation complexes.
*Model of RNA Polymerase II Transcription Initiation Machinery. The machinery depicted here encompasses over 85 polypeptides in ten
(sub) complexes: core RNA polymerase II (RNAPII) consists of 12 subunits; TFIIH, 9 subunits; TFIIE, 2 subunits; TFIIF, 3 subunits; TFIIB,
1 subunit, TFIID, 14 subunits; core SRB/mediator, more than 16 subunits; Swi/Snf complex, 11 subunits; Srb10 kinase complex, 4
http://www.bio.davidson.edu/courses/genomics/2002/james/favoriteyeastproteins.htmsubunits; and SAGA, 13 subunits. This figure
provided by Comprehensive Yeast Genome Database.
29. The activity of many promoters are greatly increased by
sequence called enhancers which can exert their
stimulatory actions over distances of several thousands
base pairs. Enhancers can be upstream, downstream or
even in the midst of transcribed gene.
Activators (gene specific transcription factors) can
provide extraboost in transcription. Activators can bind to
enhancers and also permits cells to control expression of
31. The region downstream of the polyadenylation site is essential for
Cleavage of the nascent transcript at multiple sites downstream of the
polyadenylation sites downstream of polyadenylation site is required
The transcript cleavage occurs cotranscriptionally and presumably
preceded cleavage at the polyadenylation site.
The product is immature mRNA Pre mRNA (Primary transcript).
The primary product of RNA transcription; the hnRNAs contain both
intronic and exonic sequences.
These hnRNAs are processed in the nucleus to give mature mRNAs
that are transported to the cytoplasm where to participate in protein
Eukaryotic Transcription Termination and mRNA Splicing
32. RNA Processing
The cap structure is added to the 5' of the newly transcribed mRNA
precursor in the nucleus prior to processing and subsequent transport of
the mRNA molecule to the cytoplasm. The 5' cap is a 7-methylguanosine
Step by step removal of introns and joining of remaining exons; it takes
place on a special structure called spliceosomes.
Addition of poly A tail Synthesis of the poly (A) tail involves cleavage of
its 3' end and then the addition of about 40- 200 adenine residues to form
a poly (A) tail. Poly A tail appears to increase stability of the resulting
33. RNA Processing
34. Alternative Splicing
Alternative splicing: is a very common phenomenon in higher
eukaryotes. It is a way to get more than one protein product
out of the same gene and a way to control gene expression in
The overall scheme is similar in bacteria and eukaryotes, but there are
significant difference, especially added complexity of the eukaryotic
translation initiation system.
Translation is the process by which ribosomes read the genetic message in
the mRNA and produce a protein according to message instruction.
37. The Genetic Code
The purine and pyrimidine bases of the DNA molecule are
the letters or alphabet of the genetic code.
Series of codons in part of a mRNA molecule. Each codon
consists of three nucleotides.
64 different combination of bases; 61 of them code for 20
amino acid (AA); the last 3 codon (UAG,UGA,UAA) do
not code for amino acids, they are termination codons.
The sequence of codons in the mRNA defines the primary
structure of the final protein.
Degenerate, specific, no gaps, non overlapping, almost
Factory for protein synthesis.
Composed of ribosomal RNA and ribosomal proteins;
known as a Ribonucleoprotein (RNP).
Translate messenger RNA (mRNA) to build
polypeptide chains using amino acids delivered by
transfer RNA (tRNA).
40. Ribosome Genetic Code
41. Large Ribosomal Subunit
A site bind to an aminoacyl tRNA
(tRNA bound to an amino acid).
P site bind a peptidyl tRNA (a
tRNA bound to peptide being
E site binds a free tRNA before it
is exist the ribosome.
42. Preparatory Steps for Protein Synthesis
First, aminoacyl tRNA synthetase join amino acid to their specific tRNA
begin with the activation of amino acids with AMP derived from ATP.
Second, ribosomes must dissociate into subunits at the end of each round
The protein synthesis occur in 3 phases:
Accurate and efficient initiation occurs, the ribosomes binds to the
mRNA, and the first amino acid attached to its tRNA.
Chain elongation, the ribosomes adds one amino acid at a time to the
growing polypeptide chain
Accurate and efficient termination, the ribosomes releases the mRNA and
43. Translation Initiation
The initiation phase of protein synthesis requires many
The small subunit of the ribosome binds to a site "upstream"
of the start of the message.
The small subunit of the ribosome proceeds downstream (5' -
3') until it encounters the start codon AUG.
Then the small subunit of the ribosome is joined by the large
subunit and a special initiator tRNA.
The initiator tRNA binds to the P site on the ribosome.
In eukaryotes, initiator tRNA carries methionine (Met).
Bacteria use (fMet.)
44. Translation (Initiation in Bacteria)
The initiation codon in prokaryotes is usually AUG, but it can also be
GUG, or more rarely, UUG.
Dissociation of the 70s ribosomes into 50s and 30s subunits under the
influence of IF1
Binding of IF3 to the 30S subunit, which prevents reassociation
between the ribosomal subunits.
Binding of IF2,IF2 and GTP alongside IF3.
Binding of mRNA and fMet-tRNAfMet to form the 30S initiation
complex. These two components can apparently bind in either order,
but IF2 sponsors fMet-tRNAfMet binding, and IF3 sponsors mRNA
binding. In each case, the other initiation factors also help.
Binding of the 50S subunit, with loss of IFI and IF3
Dissociation of IF2 from the complex, with simultaneous hydrolysis of
GTP. The product is 70 S complex ready to begin elongation.
45. Binding between the 30S prokaryotic ribosomal subunit and the initiation site of an mRNA
depends on base pairing between a short RNA sequence called the Shine-Dalgarno sequence
just upstream of the initiation codon, and a complementary sequence at the 3' - end of the
16S rRNA. This binding is mediated by IF3, with help from IF1 and IF2. All three initiation
factors have bound to the 30S subunit by this time.
46. Eukaryotic Initiation
Eukaryotic 40S ribosomal subunits, together with the
initiator tRNA (tRNAiMet), generally locate the
appropriate start codon by binding to 5'-cap of an mRNA
and scanning downstream until they find the first AUG in a
In 5-10% of the cases, the ribosomal subunits will bypass
the first AUG and continue to scan for a more favorable
The elongation processes in bacteria and eukaryotes are very similar
To begin elongation, another amino acid needed to join the first. The second amino acid
arrives bound to tRNA and the nature of this aminoacyl-tRNA is dictated by the second
codon in the message. The second codon is in the A site, which otherwise empty. This
step requires a protein elongation factor known as EF-TU and GTP in bacteria.
Peptide bond formation: An enzyme peptidyl transferase forms a peptide bond between
peptide in the P site and the newly arrived aminoacyl tRNA in the A site. The whole
assembly in the A site is dipeptidyl tRNA, and deacylated tRNA remains in the P site
(tRNA without its amino acids).
Translocation: the mRNA with its peptidyl tRNA attached in the A site moves one
codon's length to the left lead to; the deacylated tRNA in the P sites leaves the
ribosomes via the E sites, the dipeptidyl tRNA in the A site, along with its
corresponding codon moves into the P site. Translocation requires an elongation factor
called EF-G in bacteria ; EF-2 in eukaryotes plus GTP.
The process repeats itself to add another amino acids, and continuous over and over
until the ribosomes reaches the last codon in the message. When the polypeptide
complete , it is a time for chain termination.
Translational termination requires specific protein factors identified as
releasing factors, RFs in E. coli and eRFs in eukaryotes.
The signals for termination are the same in both prokaryotes and eukaryotes.
These signals are termination codons present in the mRNA. There are 3
termination codons, UAG, UAA and UGA.
Prokaryotic translation termination is mediated by three factors: RF1,RF2 and
RF3. RF1 recognizes the termination codon UAA and UAG; RF2 recognizes
UAA and UGA. RF3 is a GTP binding protein that facilitate binding of RF1
and RF2 to the ribosome.
Eukaryotes have two release factors: eRF1 which recognizes all three
termination codons, and eRF3, a ribosome dependent GTPase that helps eRF1
release the finished polypeptide.
Ribosomes do not release from mRNA spontaneously after termination, they
need help from ribosome recycling factor (RRF) and EF-G in bacteria.
51. Reading the instruction
means translating the code
in the mRNA from bases
building block of DNA to
55. Control of Gene Expression in Eukaryotes
prevent transcription, prevent mRNA from being synthesized.,Transcriptional
control mRNA after it has been produced.,Posttranscriptional
prevent translation; involve protein factors needed for translation.,Translational
after the protein has been producedPosttranslational,
their sequence of DNA bases.differences inare forms of the same gene with smallAlleles
noncodingDNA and/ora segment of a gene that is represented in the mature RNA product. Individual exons may contain codingn:Exo
DNA (untranslated sequences).
intervening sequence) (A noncoding DNA sequence ): Intervening stretches of DNA that separate exons.(Introns
The initial production of gene transcription in the nucleus; an RNA containing copies of all exons and introns.:Primary transcript
: RNA molecule that is not translated into a protein. Noncoding RNA genes produce transcriptscoding RNA gene-RNA gene or non
that exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA (tRNA) and ribosomal RNA
.ncRNAsand lastly longpiRNAssiRNAsand, microRNAs,snoRNAs), small RNAs such asrRNA(
are DNA elements that stimulate or depress the transcription of associated genes; they rely on tissue specific:Enhancers and silencers
binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer depending on what is bound to
Activators: Additional gene-specific transcription factors that can bind to enhancer and help in transcription activation.
(ORF): A reading frame that is uninterrupted by translation stop codon (reading frame that contains a start codonOpen reading frame
and the subsequent translated region, but no stop codon).
Directionality: in molecular biology, refers to the end-to-end chemical orientation of a single strand of nucleic acid. The chemical
convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5' end and a 3' end ( "five prime end" and
"three prime end"). The relative positions of structures along a strand of nucleic acid, including genes, transcription factors, and
polymerases are usually noted as being either upstream (towards the 5' end) or downstream (towards the 3' end).
RNA polymerase, DNA and newly formed RNA.containg: regionTranscription bubble
57. References and Further Reading
Ali Khalifa. Applied molecular biology; eds: ( Fathi Tash and Sanna Eissa). 109 pages. Egypt. University
Book Center. 2002. Available in paper copy from the publisher
Daniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. 110 pages. Washington, DC: AACC Press,
1996, ISBN 0-915274-84-1. Available in paper copy from the publisher
Daniel P. Stites, Abba T. Terr. Basic Human Immunology: 336 Pages. Appleton & Lange (November
1990). ISBN. 0838505430. Available in paper copy from the publisher
Innis, David H. Gelfand, John J. Sninsky. PCR Applications: Protocols for Functional Genomics: 566 pages.
Academic Press; 1 edition (May 17, 1999). ISBN:0123721865. Available in paper copy from the publisher
Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular
Biology of the cell. 1392 pages.Garland Science; 5 edition (November 16, 2007).ISBN. 9780815341055.
Available in paper copy from the publisher
Robert F. Mueller, Ian D. Young. Emery's Elements of Medical Genetics: Publisher: Churchill
Livingstone. 1995 ISBN. 044307125X. Available in paper copy from the publisher.
Robert F. Weaver. Molecular Biology. 600 Pages. Fourth Edition.
McGraw-Hill International Edition. ISBN 978-0-07-110216-2.
William B. Coleman, Gregory J. Tsongalis. Molecular Diagnostics. For the Clinical Laboratorian: 592 pages.
Humana Press; 4th Printing. edition (August 15, 2005). ISBN 1588293564... Available in paper copy from
Eukaryotic promoter . Internet. Available from;
Transcription factor. Available from. Fred Hutchinson Cancer Research Center
Transcription factor . Internet. Table. Available from;