Lecture 20
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Molecular biology Sabah Linjawi ١
Eukaryotic transcription factors
Eukaryotic transcription is more complex than prokaryotic transcription.
For instance, in eukaryotes the genetic material (DNA), and therefore transcription, is primarily localized to the nucleus
Where it is separated from the cytoplasm (in which translation occurs) by the nuclear membrane
DNA is also present in mitochondria in the cytoplasm and mitochondria utilize a specialized RNA
polymerase for transcription.
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Molecular biology Sabah Linjawi ٢
Eukaryotic transcription factors
This allows for the temporal regulation of gene
expression through the sequestration of the RNA in the nucleus
And allows for selective transport of RNAs to the cytoplasm, where the ribosomes reside.
The basal eukaryotic transcription complex includes the RNA polymerase and additional proteins that are
necessary for correct initiation and elongation.
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Molecular biology Sabah Linjawi ٣
Transcription Mechanisms in Eukaryotes
In eukaryotes, there are three classes of RNA polymerases: I, II and III.
Structure of the human TBP core domain complexed with DNA as determined by x-ray crystallography. The DNA includes the TATA element.
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Molecular biology Sabah Linjawi ٤
Initiation
RNA Pol II does not contain a subunit similar to the prokaryotic σ factor, which can recognize the
promoter and unwind the DNA double helix.
In eukaryotes, these two functions are carried out by a set of proteins called general transcription
factors.
The RNA Pol II is associated with six general
transcription factors, designated as TFIIA, TFIIB,
TFIID, TFIIE, TFIIF and TFIIH, where "TF" stands for
"transcription factor" and "II" for the RNA Pol II.
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Molecular biology Sabah Linjawi ٥
Initiation
TFIID consists of TBP (TATA-box binding protein) and TAFs (TBP associated factors).
The role of TBP is to bind the core promoter. TAFs may assist TBP in this process.
In human cells, TAFs are formed by 12 subunits
The transcription factor which catalyzes DNA melting is TFIIH.
However, before TFIIH can unwind DNA, the RNA Pol II and at least five general transcription factors have to
form a pre-initiation complex (PIC).
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Molecular biology Sabah Linjawi ٦
Elongation
After PIC is assembled at the promoter, TFIIH can use its helicase activity to unwind DNA.
This requires energy released from ATP hydrolysis.
The DNA melting starts from about -10 bp.
Then, RNA Pol II uses nucleoside triphosphates (NTPs) to synthesize a RNA transcript.
During RNA elongation, TFIIF remains attached to the RNA polymerase, but all of the other transcription factors have dissociated from PIC
The carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II is critical for elongation.
In the initiation phase, CTD is unphosphorylated, but during elongation it has to be phosphorylated.
This domain contains many proline, serine and threonine residues.
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Molecular biology Sabah Linjawi ٧
Termination
Eukaryotic protein genes contain a poly-A signal located downstream of the last exon.
This signal is used to add a series of adenylate residues during RNA processing.
Transcription often terminates at 0.5 - 2 kb downstream of the poly-A signal, but the mechanism is unclear.
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Molecular biology Sabah Linjawi ٨
Histone acetylation
In eukaryotes, the association between DNA and histones prevents access of the polymerase and general transcription factors to the promoter.
Histone acetylation catalyzed by HATs, which can relieve the binding between DNA and histones.
Although a subunit of TFIID (TAF250 in human) has the HAT activity, participation of other HATs can
make transcription more efficient.
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Molecular biology Sabah Linjawi ٩
The Genetic Code
Protein synthesis is based on the sequence of mRNA
Which is made up of nucleotides while proteins are made up of amino acids.
There must be a specific relationship between the nucleotide sequence and amino acid sequence.
This relationship is called genetic code, which was
discovered by Marshall Nirenberg and his colleagues in early 1960s.
It turns out that three nucleotides (a codon) code for one amino acid, as shown in the following figure.
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Molecular biology Sabah Linjawi ١٠
The Genetic Code
The standard genetic code. Synthesis of a peptide always starts from methionine (Met), coded by AUG.
The stop codon (UAA, UAG or UGA) signals the end of a peptide.
The following table applies to mRNA sequences.
For DNA, U (uracil) should be replaced by T (thymine).
In a DNA molecule, the sequence from an initiating codon (ATG) to a stop codon (TAA, TAG or TGA) is called an open reading frame (ORF)
Which is likely (but not always) to encode a protein or polypeptide.
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Molecular biology Sabah Linjawi ١١
The standard genetic code
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Molecular biology Sabah Linjawi ١٢
Order in the Genetic Code
The genetic code is not randomly assigned.
If an amino acid is coded by several codons, they often share the same sequence in the first two positions and differ in the third
position.
Such assignment is accomplished by the
design of wobble position
physical properties
The genetic code arranged according to the of amino acids
From the above figure, we see that the amino acids with close physical properties have similar codons.
Translation by tRNA
Translation is carried out by tRNA through the
relationship between its anticodon and the associated amino acid.
When a tRNA is brought to the ribosome by the pairing between its anticodon and the mRNA's codon, the amino acid attached at its 3' end will be added to the growing peptide.
In bacteria, there are 30-40 tRNAs with different anticodons.
In animal and plant cells, about 50 different tRNAs are found.
However, there are 61 codons coded for amino acids.
Suppose each codon can pair with only a unique anticodon, then 61 tRNAs would be needed
wobble position
Pairing between tRNA's anticodon and mRNA's codon.
The left figure defines the wobble position where base pairing does not obey the standard rule.
The right tables show all possible base pairings at the wobble position.
For example, guanine G can pair with both
cytosine(C) and uracil (U) ; inosine (I) can pair with cytosine, adenine and uracil.
References
Molecular Biology. P.C. Turner, A.G. Mclennan, A.D.
Bates & M.R.H. White.School of Biological Sciences, University of Liverpool, Liverpool, UK. Second edition.
BIOS Scientific Publishers, 2000.
www.web-books.com/MoBio/Free/Ch7F6.htm
en.wikipedia.org/wiki/Transcription_(genetics
www.sciencedirect.com/science/book/9780124371781
