Revison Lecture B29 Flashcards
What does the genome carry
Genetic information stored in the nucleotide sequence of the genome
Stored in the nucleotide sequnce of genome
What does the DNS nucleotide sequence determine
Amino acid sequnce of polypeptide chains and proteins
How is information of DNA transmitted
Via an intermediate rna
What si the process between
DNA –> rna
Rna –> protein
DNA – transcription –> RNA
RNA – translation –> protein
What are the components of DNA
4 bases - A C T G
Deoxyribose sugar
Phosphate
What are the bonds associated with DNA
Hydrogen bonds
Glycosidic bonds
Phosphodiester bonds
What si the structure of DNA.
Double helix
Sugar phosphate backbone
2- anti parallel chains
Run 5’-3’
What are the hydrogen bonds in DNA
A - 2 - T
C - 3 - G
What is a nucleoside
Base and sugar
What makes a nucleotide
Base
Sugar
Phosphate
What are the 2 types of sugar
Ribose
Deoxyribose
What are some RNA building blocks
ATP
CTP
GTP
UTP
What are some DNA building blocks
dATP dCTP dGTP dTTP - deoxy adenosine triphosphate
What is another name for a nucleotide
Nucleoside triphosphate
What si the enzyme used for the formation of new polynucleotide chains
DNA polymerase
What dos each incoming nucleotide do when forming a phosphodiester bond
Forms appropriate base pair with a base in the template
DNA polymerase then links this to the 3’ end of new strand
What does DNA polymerase do
Catalyses a nucleophillic attack by deoxyribose 3’OH on the proximal phosphate of the dNTP
What si the reaction of phosphodiester bond deacon fuelled by
Breaking of the high energy bonds int he dNTPs
What end does dNTPs attach to
3’ end
What is released from a phosphodiester bond
Water and pyrophosphate
What reaction is a phosphodiester reaction
Polymerisation
Consuming 2 high energy bonds
What happens to the analogue of thymidine
Incorporate into the growing viral DNA
Lacks 3’ end therefore chain elongation terminated
What does the viral reverse transcriptase have a higher affinity for
ZDV than human DNA polymerase
What are nucleosides and nucleotides also used in
Chemotherapy
What is the analogue of thymidine used for
Retrovir
What are RNA polymerases
Multimeric enzymes very large
What does ecoli have in respect to RNA pol
One RNA pol which has 5 subunits
How many eukaryotic RNA pol are there
3
What are the three eukaryotic RNA polymerases
RNA polymerase
1 - pre rRNA
2 - mRNA
3 - tRNA
What does the RNA polymerases do
They use one strand of DNA as a template
Do not require a primer
What sit he fundamental reaction in RNA synthesis
Formation of OHOSPHODIESTER bond
How does the RNA phosphodiester bond form
The 3’ hydroxyl group of the last nucleotide nucleophilically attacks the proximal phsphoryl of incoming NTP releasing pyrophosphate
What does the RNA transcription formation f the phosphodiester bond require
Magnesium in the active site of the polymerase
Reaction is thermodynamically favourable
Where does RNA transcription reactions occur
In a complex of DNA called transcription bubble
Where DNA has been unwound by approx 17bp
Explain the transcription bubble action
Moves in one direction along the DNA
DNA unwound infrint of polymerase then would behind
What way is RNA syn
5’- 3’
What is the new RNA sequnce
Complementary to template strand
Identical except Uracil instead of thymine
What are the three main stages of transcription
Initiation
Elongation
Termination
What sit he initiation step of RNA transcription
RNA polymerase binding - RNA pol bind to a specific DNA sequnce in the promoter, helped by transcription factor - protiens bound to DNA
DNA chain seperation - local unwinding of a short stretch of double stranded DNA revealing a single stranded template (3’ - 5’)
What si the elongation step of RNA transcription
Selection of correct nucleotide and catalysis of phosphodiester bond formation
Addition of nucleotides to RNA chain 5’ end for new RNA molecule is made first
What is the termination step for RNA transcription
Detect termination signals which specify which transcript ends
Release new RNA molecules
What are the 2 general typ of RNA processing
A - removal of excess sequences by the action of endonucleases and exonucleases - processing tRNA and rRNA
B - removal of excess sequences by action of endonucleases followed by rejoining of the required segments called splicing
What’s re the products of transcription of DNA
mRNA
tRNA
tRNA
Where are promoters
DNA sequences upstream in front of coding sequnce of the gene
What does the promoter do
Tell RNA pol where to start transcription and which DNA strand to transcribe
What is a TATA box
A promoter molecule
DNA sequnce 25bp upstream form start of transcription
An 8bp consensus sequenceusually entirely AT bases
Changes to this sequnce affect transcription efficiency
What si the CAAT box
Promoter element
Upstream form TATA box
Control strength of promoter signal rather than tissue/time specific expression
What si the GC box
Promoter element
GC boxes found in repeated throughout the promoter
What doe promoters bind
Transcription factors
What doe transcription factors do
Specifically recognise the DNS sequnce
Stimulate expression, some suppress, some ubiquitous
What is TFIID
General transcription factor GTF
What si TFIID needed for
RNA pol 2 than scribed genes
What is TFIID composed of
Complex of proteins
One is tbp ( TATA box binding protein)
What does TBP introduce to DNA
A kink in the DNA
Determines transcriptional start and direction
What does TFIID provide
Landing platform for further transcription factors and for RNA polymerases 2
What si the initiation of transcription for RNA pol 2
Formation of pre-initiation complex and most of the control of transcription occurs at this step
Complex contains RNA pol II and six general transcription factors including TFIID
Before transcription can start the pre initiation complex must form an open complex where RNA pol II is phosphorylated Nd the DNA strand s are sep
GTF required for all promoters and Evan has specific function
What are some GTF functions
TATA box recognition by TFIID by tbp subunit
Recruitment of RNA pol II
What sit he initiation of basal transcription
TFIID binds promoter as tbp revenues TATA box
RNA pol II and additional transcription factors bind
DNA is unwound locally to expose template 3’-5’
Open complex forms RNA pol II phsphorylated
Some tf released
RNA pol II and elongation factor extend transcript
TFIID remains promoter a new initiation complex can assemble
Pol II eventually dephos and recycled
For higher levels of expression what are required
Activators
Eg
Gene sp transcription factors
What happens in elongation
The phos RNA po II syn RNA
RNA pol II and emotion factor extend transcript
Leaving TFIID and promoter which initiate a new round of synthesis
Elongation factors stimulate the RNA synthesis
Elongation may be stopped leading to premature termination
What is Rho-dependant termination
A protien called Rho unwinds the new RNA-DNA duplex catching up with RNA pol at the termination site (where RNA pol pauses) causing enzyme to dissociate
What is Rho- indept termination
A hairpin loop upstream of s serious of U and dislodges RNA pol form the DNA template to,ringing transcription
How does RNA pol 1 stop syn
Uses a transcript terminating factor
How does RNA pol II stop syn
Sequnce AAUAAA
Once terminated the transcript is released in open form of RNA pol II released, dephos and recycled
How does RNA pol III stop syn
Uses a method similar to the Rho- indept method in procarytoes
How do procarytoes terminate
Via Rho- dept or indept termination
Sat does specific regulation of transcription require
Specific transcription factors not GTP
What are the specific regulation factors described as
Enhancers or silencers
What doe the specific transcription factors do
Either regulate transcription positive or negative
Where are the STP found
Either upstream or downstream form initiation site
What does looping do
Brings enhancer and promoter together
What so enhancers enhance
Formation of an initiation complex
What are transcription factors
DNA binding protiens
What are the 2 functional domains of transcription factors
DNA binding domain
Transcriptional activation domain
What does the DNA binding domain do in transcription factors
Interacts with DNA via major or minor groove
Helix run helix motif
Leucine zipper motif
Zinc finger motif
What does the transcriptional activation domain do in transcription factors
Stimulate the formation of an active transcription complex
May interact with the RNA pol complex
Indirect effect from interaction with coactivatirs and corepressors
May remodel chromatin
How do transcription factors bind
Suing h bond
Electrostatic attraction
Hydrophobic interactions
Trans acting factors
What si the Leucine zipper motif
Motif bind I the DNA major groove w/strong electrostatic charges between base and protein
Protein dimers form due to hydrophobic interaction of leucine rich areas
What are zinc finger proteins
1/2 zinc ions tetrahedrally coordinated with cysteine, histidine side chains
What are response elements
Enhancers which respond to detain metabolic factors
What are some examples of response elements
Heat shock element
Glucocorticoid response element
Metal response element
Cyclic AMP response element
What do response elements have bound to them
Transcription factors
What are glucocorticoid response element
Steroids transported in blood bound to albumin or specific transport proteins
How do glucocorticoid repose element get into target cell
Free steroids enter target cell by diffusion across mem
Bind to inactive steroid receptors in cyto
What happens to the glucocorticoid response when in target cell
Receptor dimerises exposing nuclear lovalising signal
Complex trans located to nucleus where bind to so DNA activating promoter
Acts as transcription factor
What does glucocorticoid repsonse element control pemit
The coordinated regulation of set of genes
What is an epi genetic mechanism
Modifications of DNA that switch sp gene expression on or off
What is chromatin structure
DNA in cell nucleus tightly packed with HISTONES so gene promoters not easy to get
Histone packing, Nucleosome stability and DNA accessibility tightly controlled by acetylation anddecactytleationg of lysine residues of core HISTONES
Some trans factors can do this themselves
What is methylation of DNA
Affects gene expression
Methylation generally associated with regions DNA less active transcribing RNA
Usually shuts off expression
Cytosine tagged with ch3 group
What does the total DNA constitue
The genome
