UNIT 3
Transcription
and
Protein Synthesis
Objectives
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Discuss the flow of information from DNA to RNA to Proteins

Explain transcription
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Differentiate introns and exons

State functions of the noncoding regions of mRNA

Describe post transcriptional modification of pre-RNA
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Distinguish between mRNA, tRNA and rRNA
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Distinguish the roles of ribosomes, tRNA and mRNA in protein
synthesis
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Give detailed description of the process and steps of translation

Describe application of SDS-Page gel electrophoresis to protein
analysis
DNA to RNA to Proteins
DNA to RNA to Proteins
Eukaryotes vs. Prokaryotes
In prokaryotes, transcription and translation are
coupled
 translation begins while the mRNA is still being
synthesized.
mRNA in Prokaryotes
DNA to RNA to Proteins
Eukaryotes vs. Prokaryotes

In Eukaryotes,
transcription and
translation are spatially
and temporally separated

transcription occurs in the
nucleus to produce a premRNA molecule.

pre-mRNA is processed
to produce the mature
mRNA, which exits the
nucleus and is translated
in the cytoplasm.
mRNA in Eukaryotes
DNA to RNA to Proteins
Eukaryotes vs. Prokaryotes
Transcription

occurs in four main stages:
1) binding of RNA polymerase to DNA at a promoter
2) initiation of transcription on the template DNA
strand
3) elongation of the RNA chain
4) termination of transcription along with the release
of RNA polymerase and the completed RNA product
from the DNA template.
Transcription
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Binding of polymerases to the initiation site at the promoter.

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Unwinding of the DNA double helix by helicase.

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Prokaryotic RNA polymerases have the helicase activity, but eukaryotic
RNA polymerases do not.
Unwinding of eukaryotic DNA is carried out by a specific transcription
factor.
Synthesis of RNA based on the sequence of the DNA template
strand.

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Prokaryotic RNA polymerases can recognize the promoter and bind to it
directly, but eukaryotic RNA polymerases have to rely on other proteins
called transcription factors.
RNA polymerases use nucleoside triphosphates (NTPs) to construct a
RNA strand.
Termination of synthesis.

Prokaryotes and eukaryotes use different signals to terminate
transcription.
Promoter

The DNA promoter region in prokaryotes is a stretch
of about 40 base pairs adjacent to and including the
transcription start point.

The essential features of the promoter are the start
point (designated +1 and usually an A), the sixnucleotide -10 sequence, and the six-nucleotide -35
sequence.

The two key sequences are located approximately 10
nucleotides and 35 nucleotides upstream from the start
point.
Prokaryotic promoter
Elongation


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During elongation, RNA polymerase binds to
about 30 base pairs of DNA
At any given time, about 18 base pairs of DNA
are unwound, and the most recently synthesized
RNA is still hydrogen-bonded to the DNA,
forming a short RNA-DNA hybrid (12 bp long,
but it may be shorter)
The total length of growing RNA bound to the
enzyme and/or DNA is about 25 nucleotides.
Termination
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
Requires a termination sequence that triggers
the end of transcription.
Two classes exist:
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rho dependent
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a short complementary GC-rich sequence (followed by
several U residues) will form a "brake" that will help
release the RNA polymerase from the template.
rho independent.

binding of rho to the mRNA releases it from the
template.
Termination
rho-dependent –
requires a protein called
rho to bind to specific
sequence.

Termination

rho-independent depends on a seqence in
mRNA which forms a
stem loop.
Termination
Transcription in Eukaryotes
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Although transcription in eukaryotes is similar to that in
prokaryotes, the process is more complex.

Instead of one RNA polymerase, there are three :
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RNA polymerase I (localized to the nucleolus) transcribes
the rRNA precursor molecules.
RNA polymerase II produces most mRNAs and snRNAs.
RNA polymerase III is responsible for the production of
pre-tRNAs, 5SrRNA and other small RNAs.
The mitochondria and chloroplasts have their own RNA
polymerases.
Transcription complex

http://highered.mcgrawhill.com/sites/0072437316/student_view0/chap
ter18/animations.html#
Transcription in Eukaryotes
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Eukaryotic nuclear genes have three classes of
promoters which are individual for the three
types of RNA polymerases
Transcription in Eukaryotes


Termination signals end the transcription of
RNA by RNA polymerase I and RNA
polymerase III without the activity of hairpin
structures as seen in prokaryotes.
mRNA is cleaved 10 to 35 base-pairs
downstream of a AAUAAA sequence (which
acts as a poly-A tail addition signal).
RNA processing in Eukaryotes

Ribosomal RNA processing

involves cleavage of multiple rRNAs from a
common precursor, with nontranscribed spacers
separate the units.

The transcription unit is transcribed by RNA
polymerase I into a single long transcript (prerRNA)
RNA processing in Eukaryotes

Every tRNA gene is transcribed as a precursor
that must be processed into a mature tRNA
molecule by:
removal of the leader sequence at the 5΄ end
 replacement of two nucleotides at the 3 ΄ end by the
sequence CCA (with which all mature tRNA
molecules terminate)
 chemical modification of certain bases
 excision of an intron.

RNA processing in Eukaryotes
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
Transcription of eukaryotic pre-mRNAs often
proceeds beyond the 3΄ end of the mature
mRNA.
An AAUAAA sequence located slightly
upstream from the proper 3΄ end then signals
that the RNA chain should be cleaved about 1035 nucleotides downstream from the signal site,
followed by addition of a poly-A tail catalyzed
by poly(A) polymerase.
RNA processing in Eukaryotes

Messenger RNA in eukaryotes

is first made as heterogeneous nuclear mRNA /
pre-mRNA then processed into mature mRNA
through:
the addition of a 5 prime cap - a guanosine nucleotide
methylated at the 7th position
 addition of poly-A tails
 the splicing out of introns.

Introns and Exons

Eukaryotic genes have interrupted coding sequences.

There are long sequences of bases within the proteincoding sequences of the gene that do not code for
amino acids in the final protein product.

The nocoding regions within the gene are called
introns (intervening sequences).

The exons (expressed sequences) are part of the
protein-coding sequence.
How introns are removed
How introns are removed



The intron loops out as
snRNPs (small nuclear
ribonucleoprotein particles,
complexes of snRNAs and
proteins) bind to form the
spliceosome.
The intron is excised, and the
exons are then spliced
together.
The resulting mature mRNA
may then exit the nucleus and
be translated in the
cytoplasm.
Animation –
How introns are removed

http://highered.mcgrawhill.com/sites/0072437316/student_view0/chap
ter15/animations.html#
Introns and Exons



A typical eukaryotic gene may have multiple
exons and introns and the numbers are quite
variable.
In many cases the lengths of the introns are
much greater than those of the exon sequences.
For instance the ovalbumin gene contains about
7700 base pairs, 1859 of them in exons.
Ovalbumin gene
Functions of Introns

Evidence now exists that introns have many
functions, including for regulation and structural
purposes, and that many of the roles now
hypothesized for introns are plausible but need
further elucidation.
Please read at least three of the
following articles for tutorial
http://www.sciencedaily.com/releases/2009/05/090528203730.htm
http://www.sciencedaily.com/releases/2007/07/070712143308.htm
http://www.sciencedaily.com/releases/2006/11/061113180029.htm
http://www.sciencedaily.com/releases/2009/06/090606105203.htm
http://www.sciencedaily.com/releases/2006/04/060404090831.htm
http://www.sciencedaily.com/releases/2005/10/051020090946.htm
http://www.sciencedaily.com/releases/2009/05/090520140408.htm
http://www.sciencedaily.com/releases/2008/11/081104180928.htm
http://www.sciencedaily.com/releases/2008/10/081017080145.htm
Roles of ribosomes, mRNA and
tRNA in Protein Synthesis
Protein Synthesis

http://highered.mcgrawhill.com/sites/0072437316/student_view0/chap
ter15/animations.html#
References/ sources of images
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http://evolution.berkeley.edu/evolibrary/article/0_0_0/mutations_03
usmlemd.wordpress.com/2007/07/14/dna-replication/
http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/centraldogma.jpg
http://www.usask.ca/biology/rank/demo/replication/cons.rep.gif
http://click4biology.info/c4b/3/images/3.4/SEMICON.gif
http://www.bio.miami.edu/~cmallery/150/gene/sf12x16.jpg
http://publications.nigms.nih.gov/findings/sept08/images/hunt_gene_big.jpg
http://ghr.nlm.nih.gov/handbook/illustrations/duplication.jpg
http://images.google.com.jm/imgres?imgurl=http://ghr.nlm.nih.gov/handbook/illustrations/duplication.jpg
&imgrefurl=http://ghr.nlm.nih.gov/handbook/illustrations/duplication&usg=__BgKRLXXosxRaUqN5EyP7qchszc=&h=400&w=370&sz=38&hl=en&start=2&tbnid=ZfARmmvAKG02xM:&tbnh=124
&tbnw=115&prev=/images%3Fq%3Dduplication%2Bmutation%26gbv%3D2%26hl%3Den%26client%3Dfir
efox-a%26rls%3Dorg.mozilla:en-US:official%26sa%3DG
http://images.google.com.jm/imgres?imgurl=http://www.phschool.com/science/biology_place/biocoach/im
ages/transcription/euovrvw.gif&imgrefurl=http://www.phschool.com/science/biology_place/biocoach/trans
cription/tctlpreu.html&usg=__hoX0ehn3x9zc2nUeciZ8gLYLqQ=&h=288&w=261&sz=20&hl=en&start=10&um=1&tbnid=X11pPXboE
KhRIM:&tbnh=115&tbnw=104&prev=/images%3Fq%3Dtranscription%26ndsp%3D18%26hl%3Den%26sa
%3DG%26um%3D1
www.asa3.org/ASA/PSCF/2001/PSCF9-01Bergman.html
http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-21/CB21.html
http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-21/2101.jpg