Week 2 (Transciption) Flashcards
How can a cell change the expression of each of its genes?
Most commonly by controlling the production of its RNA
Differences between DNA and RNA
(1) The nucleotides in RNA are ribonucleotides contain the sugar ribose rather than deoxyribose
(2) contains the base uracil (U)instead of thymine (T).
(3) RNA is single stranded
(4) individual strands of RNA are considerably shorter than that of DNA
What is the ‘transcript’?
The RNA chain produced by transcription
What are the main enzymes involved in transcription?
RNA polymerases
What is the function of RNA polymerase in transcription?
- Catalyses the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain
- RNA polymerases move stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose new region of the template strand for complementary base-paring
What are the substrates in transcription?
Where does the energy to drive the reaction come from?
Ribonuceloside triphosphates (ATP, CTP, UTP and GTP) Hydrolysis of the high energy bonds provides energy for this process
Why is RNA synthesis so efficient?
- The almost immediate release of the RNA strand from the DNA as it is synthesised means that many RNA copies can be made from the same gene in a relatively short time
- Additional RNA molecules started before the previous RNA molecules are completed
What is the main difference between DNA and RNA polymerase?
RNA polymerases can start an RNA chain without a primer
What happens if an incorrect nucleotide is added to the growing RNA chain?
The polymerase can back up, and the active site of the enzyme can perform an excision reaction that resembles the reverse of the polymerization reaction, except that a water molecule replaces the pyrophosphate an a nucleoside monophosphate is released
What are messenger RNA (mRNA) molecules
RNA molecules that are copied from genes that code for specific sequences of amino acids in proteins
What is noncoding RNA?
What are they?
Do not code for proteins
Enzymatic, structural and regulatory components
Function of rRNA?
Ribosomal RNAs
-form the basic structure of the ribosome and catalyse protein synthesis
Function of snRNA?
Small nuclear RNAs
-function in a variety of nuclear processes, including the splicing of pre-mRNA
Function of snoRNAs?
Small nucleolar RNAs
-help to process and chemically modify rRNAs
Function of miRNA?
MicroRNAs
-regulate gene expression by blocking translation of specific mRNAs and cause the degradation
Function siRNAs?
Small interfering RNAs
-turn off gene expression by directing the degradation of selective mRNAs and the establishment of compact chromatin structures
Function of piRNAs?
Piwi-interacting RNAs
-bind to piwi proteins and protect the germ line from transposable elements
Function of IncRNAs?
Long noncoding RNAs
-many of which serve as scaffolds; they regulate diverse cell processes, including X-chromosome inactivation
What is the RNA polymerase holoenzyme?
What is its function?
A sigma subunit associated with the core enzyme that contains of:
2 alpha subunits, 1 beta subunit, 1 beta prime subunit
Assists in reading the signals in DNA that tell it where to begin transcribing
What is a promoter?
A special sequence of nucleotides indicating the starting point for RNA synthesis
What is the ‘transcription bubble?’
The region of unpaired DNA
It is stabilized by binding of the sigma factor to the unpaired bases on one of the exposed strands
What happens at a terminator?
The polymerase halts and released both the newly made RNA molecule and the DNA template
The free polymerase core enzyme then reassociates with a free sigma factor to form a holoenzyme
Explain the process of transcription in prokaryotes: initiation
- RNA polymers holoenzyme binds to the DNA at the promoter to form the close promoter complex
The -35 box is bound by the sigma subunit and the rest of the enzyme sits over the -10 box and the +1 position - RNA polymerase prises the two strands of DNA apart at the -10 box which is rich in A-T base pairs and are only held together by two hydrogen bonds so is easier for the two strands to be split apart
- Sigma subunit dissociates and the core enzyme is produced and starts making RNA (first base at the +1 position) all the way through the gene
What are the 3 sections of a promoter in E.Coli.
-35 box
-10 box
+1 position
What does “-35 box “ mean?
What is it’s function?
Consensus sequence: 5’- TTGACA-3’ is 35 bases before the start point of transcription
Sequence is recognised by the sigma subunit
What does “-10 box” mean?
What is it’s function?
Consensus sequence 5’-TA TAAT-3’ is 10 sequences before the start point of transcription
Helix melting (pulling apart of the DNA strands by RNA polymerase) starts here
What does “+1 position” mean?
What is it’s function?i
It is still before the coding sequence and this is the position at which RNA polymerisation starts
What is the function of RNA polymerase I?
Synthesises rRNA
What is the function of RNA polymerase II?
Synthesises mRNA
What is the function of RNA polymerase III?
Synthesises tRNA + other small RNAs
What are the subunits if RNA polymerase II called?
Transcription factors (TFIIx)
What does eukaryote promoter consist of?
- 25 / TATA box 25 bases before the start site of transcription
Consensus: 5’-TATAAAT-3’
Explain initiation of transcription by RNA polymerase II
- TFIID: Recognises and binds to the TATA box via TBPs
- TFIIA: Binds to the DNA and stabilises this interaction
- TFIIB: Binds to the promoter assembly and accurately positions RNA polymerase at the start site of transcription
- A complex of RNA polymerase II and TFIIF binds to the assembly as they recognise and attract the other transcription factions
- TFIIH and TFII E bind thus allowing transcription to commence by prying apart the DNA double helix using energy from ATP it also changes its conformation to release polymerase from the assembly
This is the transcription initiation complex
Explain the elongation process of transcription by RNA polymerase II?
- RNA polymerase reads the template strand and match up incoming bases in the form of ribonucleotide (nucleotide triphospahtes at this point)
- RNA polymerase ensure complimentary base pairing
- RNA polymerase forms a phosphdiester bond between the alpha phosphate of the incoming nucleotide and the 3’ OH of the proceeding nucleotide with the loss of pyrophosphate (PPi)
- This repeats all the way through the coding region (Gene) and past the end of the gene until something tells it to stop
What is are important properties of terminators?
They are palindromes
Explain the elimination process of transcription in prokaryotes?
- RNA polymerase goes past the end of the coding sequence then transcribes the palindrome
- The palindrome in the RNA folds intoas stem loop
- The stem loop entangles the RNA polymerase which causes the polymerase to pause and complex dissociates and RNA is released
- DNA base pairs back together
Name the two ways in which the complex dissociates during termination
Rho dependent
Rho independent
Describe Rho independent dissociation
A series of As and Us are transcribed after the stem loop so the base pairing between the RNA and the DNA is weak
Difference between prokaryotic mRNA and eukaryotic mRNA?
Eukaryotic mRNA isn’t continuous, it has to be spliced
Eukaryotic mRNA is made in the nucleus and translated in the cytoplasm
Why does pre- RNA need to be spliced?
Because it contains copies of both introns and exons and the introns need to be removed
What are the 3 stages of pre-mRNA processing?
- Capping
- Polyadenylation
- Introns splicing
what is Capping?
When the RNA polymerase II has produced about 25 nucleotides of RNA, the 5’ end of the new RNA molecule is modified by the addition of a cap that consists of a modified guanine nucleotide.
- G attached in 5’-5’ bond
- Cap G (modified guanine is methylated at the 7 position
- next two bases can also be methylated
What is Polyadenylation?
- Sometime after the end of the coding sequence and before the palindrome, there will be a polyadenylation signal
- Consensus sequence: 5’AAUAAA-3’
- Enzymes recognise this sequence and they cleave the mRNA transcript 10-30 bases downstream from the polyadenylation signal
- poly(A) polymerase adds up to 250 As on the 3’ end of the RNA
- terminator sequence is cut off
What is intron splicing?
- Joins exons to form whole gene
- Eukaryotic pre-mRNA has GT-AG class introns
What are snRNPs and what do they do.
Small nuclear ribonuclear proteins
RNA + proteins together that cut out the introns
Explain intron splicing in detail
- The intron is cut at the 5’ end just before GU bases by snRNPs and it forms a 5’ to 2’ bond with the branch A
- The snRNPs make a cut at the other end of the intron (after AG bases)
- Intron released as a lariat (lasso)
- Joins the two exons together
What are the 3 main types of RNA?
mRNA: messenger RNA (translated into a polypeptide
rRNA: ribosomal RNA (end point of gene expression)
tRNA: transfer RNA (end point of of translation)
What is the function of rRNA in the ribosome?
- Forms a molecular scaffold for peptides
- Involved in catalytic activity
What does the s of ribosomal subunits stand for?
Svedberg
What is the Svedberg a measurement of?
How the ribosomal subunits behave when centrifuged in a sucrose density gradient
What is the measurement of the small and large subunits of prokaryotic ribosomes?
Large subunit: 50 s (34 peptides + 2 RNA)
Small subunit: 30 s (21 peptides + 1 RNA)
70s
What is the measurement of the small and large subunits of eukaryotic ribosomes?
Large subunit: 60 s ( 49 peptides + 3 RNAs)
Small subunit: 40 s ( 33 peptides + 1 RNA)
80s
How does E.Coli make enough rRNA in the correct proportions?
It has 7 copies of the 3 genes and they are transcribed together
This produces pre rRNA which go on to be processed
How many copies of the rRNA do eukaryotes have?
How many are transcribed together
Between 50-5000 of 4 genes
3 are transcribed together > pre rRNA
How is the mRNA read?
In 3 base units (codons)
Each codon encodes 1 amino acid
How does the tRNA read the mRNA?
Via the anticodon
How does tRNA base pair with mRNA?
In an antiparallel fashion
Difference between codon and anticodon
Codon - In mRNA
Anticodon- In tRNA
What is wobble
Flexibility (special rules) for the base pairing at the third position of the codon/ first position at the anticodon
What other base pairing rules does wobble entail.
G=U
I=A
I=C
I= U
What is the modified base that occurs in tRNA
Inosine
Describe the synthesis of mature tRNAs
- transcription of several tRNA genes > pre tRNA
- tRNA has sequences that can base pair with themselves which causes them to fold
- tRNAs are spliced from the transcript by RNase P at the 5’ end and RNase D at the 3’ end
- Some bases are modified enzymativally
- Aminoacylation occurs (amino acid attached by the enzyme tRNA synthetase) also called charging which required energy in the form of ATP to AMP with the loss of PPi (pyrophosphate)
What is the challenge for amino acyl tRNA synthetases (AARS) ?
Multiple codons for most amino acids
They have to cope with binding multiple tRNAs for the same amino acid
Which RNA will thymine only occur in?
tRNA (uracil modified to make Ribothymidine)
Why could the chemical modification of tRNA be useful?
- Help AARS to recognise specific tRNA
- contribute to the chemistry at the wobble position
Explain the process of aminoacylation
- Amino acyl tRNA synthetase binds to the correct amino acid
- Binds to ATP, forming a covalent bond between the amino acid and AMP and the correct tRNA
- A complex is formed ( AARS + AA + AMP + tRNA
- AARS enzyme catalysed a link between the AA and the tRNA
- The charged tRNA is released
Last 3 bases 5’-CCA-3’
What is a consensus sequence?
The most common nucleotide found at each position in the promoter
What is the direction of transcription determined by?
The promoter at the beginning of each gene
What are TBPs?
TATA binding proteins
A subunit of TFIID that is responsible for recognising and binding to the TATA box sequence in the DNA
Which transcription factor contains a DNA helicase?
TFIIH
What conformation changes occur that allows DNA polymerase to move away from the promoter?
Addition of phosphate groups to the ‘tail’ of the RNA polymerase know as the CTD (C-terminal domain)
Name another function of TFIIH
What does it contain in one of its subunits
during transcription initiation, the serine located at the 5th position in the repeat sequence is phosphorylated by TFIIH which contains a protein kinase in one of its subunits
What are elongation factors?
How do histone chaperone help?
proteins that decrease the likelihood that that RNA polymerase will dissociate before it reaches the end of the gene
Histone chaperone help by partially disassembling nucleosomes in front of a moving RNA polymerase and assembling them behind
Describe superhelical tension in relation to RNA polymerase elongation
Supercoiling is a conformation that DNA adopts in response to superhelical tension .
RNA polymerase creates superhelical tension as it moves along DNA, positive superhelical tension in front of it and negative behind it
What is the function of DNA gyrase?
- The specialised bacterial topoisomerase uses energy from ATP hydrolysis to pump supercoils continuously into the DNA thereby maintaining the DNA under constant tension
- DNA gyrase therefore makes the opening of the DNA helix in bacteria energetically favourable compared with helix opening in the DNA that is not supercoiled
How and why are both ends of eukaryotic mRNA modified?
- Capping on the 5’ end and by polyadenylation of the 3’ end.
- These special ends allow the cell to access whether both of an mRNA molecule are present (and if the message is therefore intact) before translation occurs
What is the purpose of phosphorylation at the CTD?
- Helps dissociate the RNA polymerase II from other proteins at the start point of transcription
- Allows a new set of proteins to associate with the RNA polymerase tail that function in transcription elongation and RNA processing
Describe the role of the 3 enzymes involved in RNA capping
- Phosphatase removes a phosphate from th e5’ end of the nascent RNA
- Guanyl transferase adds a GMP in a reverse linkage 5’-5’
- Methyl transferase adds a methyl group to guanosine
What is the function of the 5’ methyl cap
Helps the cell distinguish mRNAs from other types of RNA molecules
Where does wobble occur
Between the third base of the codon and the first base of the anticodon
Is the first base of the anticodon affected by wobble?
Yes
Is the third base of the codon affected by wobble?
No
How is ribothymidine produced?
Methylation of uracil
When is Rho needed?
When the final region is not AT rich
Where does transcription in prokaryotes take place?
In the cytoplasm
What are the 3 steps of pre-mRNA processing?
- Capping
- Intron splicing
- Polyadenylation
