Transcription in Prokaryotes and Eukaryotes Flashcards
What is the nucleoiod
The bacterial ‘equivalent’ (sort of) of the nucleus
NOT membrane bound
Contains DNA and proteins (HU and H1)
In prokaryotes: DNA is arranged how?
DNA is assembled in loops
Supercoiling allows DNA to coil up on itself
Loops in bateria - 10-50 kb
Loops contain DNA that is condensed by interacting with basic (positively charged proteins). DNA is negatively charged so interacts well
What is Transcription
Synthesis of RNA from a DNA template
RNA polymerase copies one strand of DNA into RNA
RNA is identical to coding strand (but U replaces T) and coplimentary to the template strand
What is a gene
Region of double stranded DNA that codes a peptide chain (protein) (OR functional RNA)
True or False?
All genes encode proteins
False
Genes encode tRNA and rRNA (involved in translation to produce proteins)
Some genes encode RNAs to help process mRNA
Some genes encode RNAs that regulate other genes
Why is the translational start codon (AUG) not found exactly at the start of the mRNA
The region before AUG does not encode protein called an untranslated region or UTR
RNA is always synthesised in which direction
What is the length of mRNA relative to the protein coding regions
5’ to 3’ direction
mRNA is longer than the protein coding regions because of the 5’ and 3’ untranslated regions
What is the Promoter
is the control region found at the start of the gene
The point where transcription starts is within the promoter
The promoter is a key control region for controlling transcription in the gene
Relative to the promoter and terminator, which way is mRNA synthesised
in the direction from promoter to terminator
i.e. information flows through the gene from left to right
Anything in the direction of the promoter is said to be …..
Upstream (against the flow of RNA polymerase)
Anything towards the terminator is …..
Downstream (with the flow of the RNA polymerase)
In bacteria, genes are often clustered together with several genes transcribed from one promoter
This is called what?
An operon
Genes whose products have related functions are often what?
Arranged together
This is however very unusual to occur in eukaryotes
What is polycistronic mRNA
Codes more than one protein
In prokaryotes
How are common signals in the DNA sequence (conserved elements) identified
100s of different promoters from different genes were aligned
For each gene, transcription always starts at a particular positon called
the promoter
RNA polymerase does what
It has how many types of subunit?
Synthesises RNA
4 types of subunit
What is the consensus sequence
Most common bases observed at these areas
A -10 sequence is relatively rich in AT base pairs which allow what
Stands to seperate more easily
(2 hydrogen bonds instead of 3)
Is or isn’t there variation within the -35 and -10 sequences
Yes there is variation
How does RNA polymerase interact with the promoter region
RNA polymerase binds to the -35 sequence
Closed complex forms over the promoter region
and unwinds the -10 sequence by melting which turn complex to open form
The promoter region has 3 componenets
What are they?
Describe the main features of RNA sysnthesis in prokaryotes
One strand used as a templated
Transcription 5’-3’ (like DNA replication)
No primer needed
The first ribonucleotide has a what
What bonds are formed between the ribonucleotides
triphosphate at 5’ end (usually an A base)
Forms a phosphodiester bond in the same way as DNA synthesis
Describe the transcrption process
- Template recognised: RNA polymerase bonds to duplex DNA and DNA is unwound at the promoter
- Initiation: Several abortive attempts are made to get going (makes short chains and then releases them) until eventually
- Elongation Polymerase synthesises RNA of a certain minimum size that is stably associated with a template DNA which it then goes on to elongate
- Termination: RNA polymerase and RNA are released
Describe how RNA moves through RNA polymerase
DNA is unwound in front, template DNA passes through active site and RNA is synthesised then passes out the back
At the active site, there is a hybrid of newly synthesised RNA and DNA (9bp)
This is why RNA polymerase takes time to get going as it needs to have something longer than 9 nucleotides to get started
Describe the coupled transcription and translation within bacteria
As RNA polymerase moves through the gene making the mRNA, ribosomes latch onto the mRNA to make protein at the same time before mRNA sythesis (transcription) completed
Makes process very fast (relative to eukaryotes because once mRNA is formed in the nucleus it must then move to the cytoplasm)
Transcription terminated at specific signals in the DNA called terminators.
What are the two types of terminators
- Intrinsic terminators
- Rho dependent terminators
- Both are 50:50 in bacteria
What are intrinsic terminators
What is the two features which it has
- All information to bring about termination is in the gene itself. Bacterial termination occurs at a discrete site. All sequences required for termination are in transcribe region, leading to RNA polymerase and RNA being released.
- Loop and stem
During intrisnsic termination newly synthesied mRNA folds into a hairpain structure, How?
A hairpin shape is formed as part of coiling over through strong G-C bond
Forms a stem loop strucutre
Causes process to stop
Single stranded U run setion which are very weakly bonded between DNA and RNA and enzymes to fall off
What causes Rho factor-dependant termination
Rho is a hexameric RNA binding protein
Recognises a rho utilisation (RUT) site in the newly synthesised mRNA
Rho-dependent terminator sequence (RUT site) rich in C and poor in G
Forms a signal in the RNA which the Rho factor can interact with
What is the process of Rho-dependent termination
- RNA polymerase transcribes the DNA incorperating the rut site into the RNA
- Rho factor recognises the rut site and interacts with RNA
- It translocates along the RNA towards the RNA polymerase
- RNA polymerase pauses at hairpin structure (similar to intrinsic terminator)
- Rho cataches up with RNA polymerase and unwinds DNA and RNA hybrid at which point everything is released and transcription is terminated
Eukaryotes: Describe the packaging of DNA
DNA is tightly packaged to fit in nucleus of cell ( has profound implications for transcription)
Packaging of DNA is done by organising FNA into chromatin (DNA + histone) using strucutres called nucleosomes
Eukaryotes: Core nucleosomes contains what?
Core nucleosome contains 2 copies of H2a, H2B, H3 and H4 - DNA wound round outside of core nucleosome
H1 associated with DNA between nucleosomes - linker histone ‘beads on a string structure’
How can 10nm Chromatin fibres condense further
nucleosome string coils around itself - solenoid forming a 30nm fibre
Eukaryotic genes are often interupted by what?
How are they removed
Introns
Introns are transcribed from DNA into RNA but removed by a processing reaction to generate the final mRNA
Eukaryotes: Which form of RNA polymerase transcribes DNA (if the gene is a protein-coding gene)
RNA polymerase ll
What is the polyadenylation site
Marks end of mRNA product
What is RNA processing reactions
Introns removed - extra regions at the 3’ end back to the polyadenylation site as well as the introns
Exons joined together to produce the final mRNA
Known as RNA splicing
Only exons are preset in the final mRNA
What is the primary transcript
Contains introns as well as extra sequence at the 3’ end (beyond the polyadenylation site)
What is the protein start codon
ATG
not the same as the RNA start point
What are the three protein stop codons
TAG
TAA
TGA
Why is the process of transcription and translation slower in eukaryotoes than it is in prokaryotes
Transcription occurs in the nucleus and translation occurs in the cytoplasm so mRNA must move through nuclear membrane making process slower - eukaryotes
In prokaryotes you get couples transcription and translation
What are the 5 different transcriptional machinery in eukaryotes and their role
RNA polymerase l - synthesis rRNA
RNA polymerase ll - synthesis mRNA
RNA polymerase lll - synthesis tRNA, snRNA, 5SRNA
General transcription factos - promoter location
Activator proteins - stimulation of transcription
RNA polymerase ll transcribes what
Protein coding genes (has 10+ subunits)
Needs general transcription factors and activator proteins to find promoters and to aid transciption at a higher level
What is the core promoter
Region where transcription starts. Contains RNA initiation site (start site) and in some core promoters there is a TATA box 25bp upstream of the RNA start site
What is the proximal promoter
next 100-200 bp upstream of the RNA start site
What is the enhancer
Often many 1000s of bases upstream - plays a role in regulating transcription from the core promoter
The DNA elements are recognised by proteins (transcription factors and activators) that target RNA polymerase ll (this polymerase cannot bind without help)
What are general transcription factors
Interact with DNA at the core promoter
Present in all cell and forms the transcription ‘machine’ in combination with RNA polymerase
What are activators
Interact with DNA and proximal promoter and enhancer
Maybe tissue specific. Regulate the level and timing of transcription of indvidual genes
Where ar the enhancers, activators, proximal and core promoters and transcription factors relative to another on DNA
General transcription factors assemble a transcription complex at the core promoter
This allows RNA polymerase II to associate
Known as a STEPWISE ASSEMBLY model
General transcription factos are called what
TFIIA
TFIIB
TFIID
TFIIE
TFIIF
where TF = transcription factor
II = RNA polymerase II associated
Which is the first transcription factor to bind
Why is the order the transcription factors bind important
First transcription factor to bind is TFIID (mix of proteins - TBP and several TAF proteins)
TBP binds in minor groove of DNA and brings TFIID into the core promoter
Followed by TFIIA
Assemble in a defined order on the core promoter to establish the transcription complex (TC)
Activators influence what?
the assembly of the transcription complex and strucuture of chromatin
They work in two ways: make assembly of the transcription complex more efficient and unpack chromatin to allow transcription
Activators are a long way from the promoter. How therefore can they react with the promoter
One model suggests they interact and influence the core promoter is by the DNA between the enhancer and core promoter looping around so the proteins attached to the enhancer make contact with the general TFs assembling at the core promoter
What are Hemingway cats
They have 6 rather than 5 toes
Results from mutation in an enhancer that regulates expression of a gene called sonic hedgehod involved in development
How does the process of eukaryotic mRNA being capped work
Capping of the 5’ end of the Eukaryotic DNA
Adds a 7-methyl Guanine Ribose to the 5’ end of the mRNA (modified G nucleotide)
Added on by an unusual 5’ to 5’ triphosphate bond
Why is mRNA capped
- The cap protects the 5’ end of the mRNA from degradation and is important for mRNA stability
- The cap interacts with translation factors which recruit ribosomes for protein synthesis
- The cap interacts with the cap binding complex involved in export of mRNA from the nucleus
- The cap may be important in splicingg introns near the 5’ end of the mRNA
Polyadenlylation occurs at the 3’ end of mRNA
How
At the 3’ end, transcription goes past the position of the end of the mRNA to make a longer RNA, which is the cut and then polyadenylated
The sequence AAU AAA in the mRNA targets a protein complex
This complex cuts the mRNA 11-30 bases past the AAU AAA
The removed part of the RNA is degraded and the remaining mRNA is polyadenylated
The poly A tail is important in determining mRNA stability and aiding translation
