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Gene Expression
Transcription and Translation
110
Biochemistry
Undergraduate 1
12/24/2012

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Term
What is the order of sequence from DNA to protein?
Definition
DNA - > Transcription -> RNA -> Translation -> Protein
Term
What is the bacterium model organism?
Definition
E. coli
Term
Why study E. coli?
Definition
1)Provides a framework for understanding more complex organisms
2)Provides an understanding of bacteria as pathogens
3)Easy to manipulate and take up recombinant DNA
4)See how antibiotics affect bacteria
Term
How many strands does DNA have?
Definition
Two
Term
How many strands does RNA have?
Definition
One
Term
What are the four RNA's?
Definition
rATP, rCTP, rGTP and rUTP
Term
What are the three regions of a gene?
Definition
Promoter sequence, gene region and terminator sequence
Term
Where are the two consensus sequences?
Definition
At -35bp and -10bp
Term
How many base pairs are between the -35 and the -10 consensus sequences?
Definition
Between 16-19bp
Term
How many base pairs are between the -10 and the start-site sequences?
Definition
Between 5-8bp
Term
What is the normal sequence of bases that make up the -35 consensus sequence?
Definition
TTGACA
Term
What is the normal sequence of bases that make up the -10 consensus sequence?
Definition
TATAAT
Term
How consensus sequences determined?
Definition
Looking at the most common base at each of the positions of a number of promoters
Term
Which ion is required for RNA Polymerase to work?
Definition
Mg2+
Term
Which components make up the Core RNA Polymerase?
Definition
2 x alpha
1 x beta
1 x beta'
1 x omega
Term
Which factor is also required for RNA Polymerase to work?
Definition
Sigma Factor
Term
What two components make up the Holoenzyme?
Definition
The Core RNA Polymerase AND the sigma factor
Term
How many domains are in the sigma factor?
Definition
Five
Term
What does the 1.1 domain of the sigma factor do?
Definition
Prevents binding to promoters in the absence of RNA polymerase
Term
What do the 1.2, 2.2, 2.3 and 2.4 domain of the sigma factor do?
Definition
Mediate binding to the -10 consensus sequence
Term
What do the 4.1 and 4.4 domain of the sigma factor do?
Definition
Mediate binding to the -35 consensus sequence
Term
What number is the main sigma factor?
Definition
70
Term
What other number sigma factors also exist and are sometimes used?
Definition
32 and 54
Term
What to 32 and 54 sigma factors mediate?
Definition
Binding to other promoter seqeunces
Term
What happens during the initiation stage of transcription?
Definition
The sigma factor binds the RNA polymerase and the strand of DNA is opened up
Term
What happens during the elongation stage of transcription?
Definition
The sigma factor leaves the RNA polymerase and the RNA polymerase transcribes the anti-sense strand using complimentary base pairing
Term
In what direction does RNA polymerase read DNA?
Definition
5' to 3'
Term
How many nucleotides can be transcribed per second in E.coli?
Definition
20-50
Term
Does RNA Polymerase have a proof reading function?
Definition
No
Term
What is the error rate of RNA Polymerase?
Definition
1/10,000
Term
What are the two types of termination?
Definition
Factor Independent and Rho dependant
Term
How does Factor Independent termination occur?
Definition
The palindromic GC sequence causes a hairpin loop to form that reduces the RNA Polymerases' affinity for the strand and so is pulled off
Term
What are the two common features of factor independent terminator strands?
Definition
1)Series of 4-10 consecutive A-T base pairs
2)A G and C rich region that is palindromic before the A/T region
Term
What is a palindromic sequence?
Definition
Opposite base pairing on the strand strand
Term
What makes up the structure of the Rho factor?
Definition
6 identical subunits
Term
How the Rho factor powered?
Definition
By nucleoside triphosphates
Term
What is the Rho factor?
Definition
A helicase enzyme that unwinds the DNA/RNA complexes
Term
How does the Rho factor cause termination?
Definition
It moves quicker than the RNA polymerase along the strand behind it. Eventually it catches up and pushes of the RNA polymerase terminating the strand
Term
How do cells use transcription to react to changes in the environment?
Definition
Altering the sets and levels the genes are expressed
Term
What are the two strategies for regulating transcription initiation?
Definition
Repression and Activation
Term
How does repression work as a strategy for regulating transcription initiation?
Definition
Repressor protein blocks the RNA polymerase and will not activate RNA polymerase until it is removed
Term
How does activation work as a strategy for regulating transcription initiation?
Definition
RNA polymerase is bound to promoter but will not begin transcription unless another protein allows it to begin transcribing
Term
What are the gene regions along the lac operon?
Definition
PlacI, lacI, Plac, O1, lacZ, lacY, lacA
Term
What does PlacI do in the lac operon?
Definition
Acts as the promoter sequence for the lacI gene
Term
What does lacI do in the lac operon?
Definition
Contains the gene for the lac repressor
Term
What does Plac do in the lac operon?
Definition
Acts as a promoter for the whole lac operon
Term
What does O1 do in the lac operon?
Definition
Acts as a binding site for the lac repressor that will prevent transcription
Term
What does lacZ do in the lac operon?
Definition
Contains the gene for B-galactosidase
Term
What does lacY do in the lac operon?
Definition
Contains the gene for lactose permease
Term
What does lacA do in the lac operon?
Definition
Contains the gene for acetylase
Term
Transcription of the lac operon only occurs under which conditions?
Definition
Abscense of glucose AND presence of lactose
Term
How many amino acids are present in the lac repressor?
Definition
360
Term
What type of molecule does the lac repressor form?
Definition
A homotetramer
Term
What is a homotetramer?
Definition
A molecule made up of four identical sub-units but not covelantly bonded
Term
Why is the lac repressor a homotetramer?
Definition
Because secondary operator sites (O2 and O3) are also present and these must also be blocked
Term
What is the main inducer for the lac operon?
Definition
Allolactose
Term
What is the alternative inducer for the lac operon?
Definition
Other B-glactosidases like IPTG
Term
How does allolactose induce the lac operon?
Definition
Allolactose binds to the repressor protein which reduces the proteins affinity for the DNA so the repressor protein leaves
Term
Outline how glucose induces transcription of the lac operon?
Definition
1)No glucose results in the conversion of ATP into cAMP
2)cAMP then binds to the CAP protein which starts transcription
Term
How does the CAP protein initiate transcription?
Definition
It binds to the RNA polymerase activating it
Term
In conditions of glucose and no lactose what occurs in terms of the lac operon?
Definition
Repressor protein present, CAP not present so no transcription
Term
In conditions of no glucose and no lactose what occurs in terms of the lac operon?
Definition
CAP present but RNA polymerase still blocked by repressor protein so no transcription
Term
In conditions of glucose and lactose what occurs in terms of the lac operon?
Definition
CAP not present but no repressor protein so a little transcription occurs
Term
In conditions of no glucose and lactose what occurs in terms of the lac operon?
Definition
CAP present and no repressor protein so transcription occurs
Term
Which organism requires a CAP protein in order to regulate transcription for toxins?
Definition
Vibrio cholerae
Term
What is the genetic code?
Definition
Sequence of four bases that codes for 20 amino acids
Term
What are the features of the genetic code?
Definition
Triplet code, degenerate and non-overlapping
Term
Is the genetic code universal?
Definition
Almost
Term
How many nucleotides make up a tRNA?
Definition
70-90nts
Term
How many areas are invariant in a tRNA molecule?
Definition
15
Term
How many areas are semi-variant in a tRNA molecule?
Definition
8
Term
How many phosphate groups do each nucleotide or tRNA have?
Definition
One
Term
What are the four modified bases in tRNA?
Definition
Ribothymine, pseudouridine, hydrouridine and inosine
Term
What are the five features of a molecule of tRNA?
Definition
D-Loop, T-Loop, variable arm, anti-codon loop, amino acid acceptor site
Term
Which part of a tRNA contains dihydrouridine?
Definition
The D-Loop
Term
Which part of a tRNA contains pseudouridine?
Definition
The T-Loop
Term
A tRNA joined to an amino acid is known as a?
Definition
Aminoacyl tRNA
Term
Which enzyme catalyses the reaction that joins a tRNA molecule to an amino acid?
Definition
tRNA synthetases
Term
Outline the mechanism that creates an aminoacyl tRNA
Definition
1)AMP is added to the carboxyl group on the amino acid to form an intermediate compound called aminoacyl adenylate
2)The aminoacyl adenylate then reacts with the tRNA to form an aminoacyl tRNA
Term
Which bond (aminoacyl or peptide) has the higher energy?
Definition
The aminoacyl bond
Term
How many different types of aminoacyl tRNA are they?
Definition
Four
Term
What are the different types of aminoacyl tRNA?
Definition
a, a2, a4 and a2b2
Term
How does the aminoacyl tRNA synthetase know which aminoacyl tRNA to use?
Definition
It looks at the identity elements
Term
What two sites do aminoacyl tRNA synthetases have that read the identity elements?
Definition
The activation site and the editing site
Term
What does the activation site do?
Definition
Reject aminoacyl tRNA's that have amino acids bigger than the correct one
Term
What does the editing site do?
Definition
Cleaves amino acids that are smaller than the correct one?
Term
Using the activation and editing sites on the aminoacyl tRNA synthetases means that a mistake is made once in every?
Definition
10,000 amino acids
Term
How does tRNA act as an adaptor between the protein and the mRNA?
Definition
The anticodon reads each codon on the mRNA which then releases the amino acid to form a peptide bond with the rest of the protein
Term
What is the Wobble hypothesis?
Definition
The first two base pairs of the anticodon match up with Watson-Crick base pairings but the third does not have to follow that rule
Term
If the base A is in the third position of the anti-codon it can/must be paired with?
Definition
U
Term
If the base C is in the third position of the anti-codon it can/must be paired with?
Definition
G
Term
If the base U is in the third position of the anti-codon it can/must be paired with?
Definition
A OR G
Term
If the base G is in the third position of the anti-codon it can/must be paired with?
Definition
C OR U
Term
If the base I is in the third position of the anti-codon it can/must be paired with?
Definition
A OR C OR U
Term
The standard ribosome size is known as?
Definition
70s
Term
What is the size of the large sub-unit?
Definition
50s
Term
What is the size of the smaller sub-unit?
Definition
30s
Term
What are the requirements for a ribosome to initiate initiation?
Definition
Initiation factors (IF1, IF2, IF3 and GTP)
A tRNAfMet
Term
What are the requirements for a ribosome to initiate elongation?
Definition
Elongation factors (Ef-Tu, Ef-Ts, Ef-G and GTP)
Term
What are the requirements for a ribosome to initiate termination?
Definition
STOP codon and release factors (RF1 and RF2)
Term
Outline the mechanism of translation initiation?
Definition
1)30s sub-unit binds to binding site after recognising the Shine-Dalgarno sequence
2)The initiator tRNA binds to start codon AUG
3)50s subunit binds to 30s sub-unit to produce the 70s ribosome complex
Term
What is the Shine Dalgarno sequence?
Definition
The sequence recognised by the 30s sub-unit to begin transcription
Term
Outline the role of the initiation factors in assembling a ribosome?
Definition
1)GTP binds to IF2 and thsi attaches the 30s sub-unit to the binding site
2)IF3 is released as initiator tRNA is bound
3)IF2 and IF1 are released when the 50s sub-unit binds
Term
Which elongation factors create a peptide bond?
Definition
Ef-Tu, Ef-Ts and GTP
Term
Which elongation factors release the tRNA body?
Definition
Ef-G and GTP
Term
The peptide bond formation is catalysed by which enzyme?
Definition
23S rRNA
Term
What does RF1 do?
Definition
Recognises UAG and UAA STOP codons
Term
What does RF2 do?
Definition
Recognises UAA and UGA STOP codons
Term
What does RF3.GTP do?
Definition
Aids RF1 and RF2
Term
What does RRF do?
Definition
Releases the ribosome from the peptide
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