Gene Expression Flashcards
What is the purpose of transcription?
Convert DNA to RNA. First step in protein synthesis
What is transcription performed by?
RNA polymerase
Describe the stages of transcription.
- Uses DNA template
- Ribonucleotides
- Produces RNA copies known as transcripts
- RNA polymerase binds to regions of DNA called promoters
- Transcribes a region before terminating
What are the subunits of RNA polymerase responsible for?
Binding to the promoter. Separating DNA strands. Transcript relocation.
What is the promoter region?
Contains sequences which are recognised by RNA polymerase.
Where are most prokaryotic promoters conserved sequences?
2 regions: -10 and -35
Where is the start of transcription?
+1. Anything before this is negative and referred to as upstream
Where is downstream?
Anything after +1
In prokaryotes what is the sigma factor involved in?
Binding to these sequences and recruiting the rest of RNA polymerase enzymes
What happens in the Promoter Melting process?
DNA strands are separated.
Which stranded of DNA is copied?
The template strand
Which direction does RNA polymerase move in?
3’ to 5’ direction with base pairing rules.
What is meant by the transcription bubble?
Only a short section is single stranded. Transcript only paired a few bases at time.
What are the Watson-Crick rules?
Complementary base pairing.
When does transcription end?
Termination signals are encountered and RNA polymerase dissociates
What do most prokaryote terminus sequences involve?
Inverted repeat structures
What happens at the terminus sequence in prokaryotes?
- Region transcribed and RNA formed will create a hairpin structure
- Causes RNA polymerase to pause and transcript factor is released
- Another factor there will be an AT rich region which when transcribed will be an AU rich region
- Will help release as weaker hydrogen bonds than CG
What does mRNA do?
Codes for proteins
What do non-coding proteins do?
Regulatory. Structural.
Describe mRNA
- Protein coding region = open reading frame
- Start and stop codon
- Surrounded by untranslated regions
How many proteins do transcripts code?
Can code for multiple proteins - typical for genes in same metabolic pathway
How can transcripts code for multiple proteins?
- Genes are clustered and therefore transcripts have multiple reading frames
What is meant by polycistronic?
Multiple open reading frames and code for multiple proteins
Why is eukaryotic mRNA more complicated than prokaryotic mRNA?
Undergoes specialised processing events at their 5’ and 3’ ends
What happens at the 5’ end on eukaryotic mRNA?
Modified by addition of 5’cap. Modified base 7-methyguanosine
What happens at the 3’ end on eukaryotic mRNA?
Addition of 150-200 adenine nucleotide (polyAtail)
Where does transcription take place?
Nucleus
What do the modifications of mRNA help with?
Ensuring RNA stability, export from nucleus and translation
What does mRNA undergo in higher eukaryotes?
Splicing
What is the purpose of translation?
RNA to polypeptides
How is A U C G translated into amino acid chains?
By ribosomes and tRNA
How is mRNA read?
Read in triplets. Each triplet is either a start, stop or an amino acid.
What is the genetic code?
- 64 codons
- Degenerate
- Often 3rd base isn’t important
- Unambiguous
- Universal
What is tRNA?
Transfer RNA
What is the role of tRNA?
- Amino acids attached to a specific tRNA
- tRNA with amino acid attached is described as charged
- Has an anticodon loop, can base pair with a codon through its anticodon
- 5’ end of codon pairs with the 3’ end of the anticodon
- Need to have the correct amino acid attached to the tRNA
What attaches amino acids to tRNA?
tRNA synthetase
Describe the tRNA synthetase.
2 binding sites: amino acid and tRNA. Reaction uses ATP, attaches them
What is the site of protein synthesis?
Ribosome
Describe a ribosome.
- Composed of RNA and proteins
- Contains a small and a large subunit
- Ribosomal RNAs provide the catalytic function for translation
What are the 3 important sites on the ribosome?
Aminoacyl site. Peptidyl Site. Exit site.
What is the role of Aminoacyl site?
Decoding centre. New charged tRNA will enter the ribosome. Anticodon and codon will pair here.
What is the role of Peptidyl site?
Growing polypeptide chain is located
What is the role of Exit site?
tRNAs released when no longer needed
Where are the A and P site located?
Next to each other so peptide bonds can form
What is the start codon?
AUG
What is translation initiation in Prokaryotes?
- Shine Dalgamo sequence which is located upstream of the start codon in the 5’ untranslated region
- Shine dalgarno is able to pair the 165 ribosomal RNA - small subunit
- Anchors the mRNA so that the start codon is in the P site
- Modified version of the methionine is used for the initiating amino acid
What is translation initiation in Eukaryotes?
- Small subunit and initiating tRNAiMet binds to the 5’cap
- Then scans the mRNA until the first AUG is reached in the P site
Describe the stages of Translation Elongation
- A ‘charged’ amonacyl tRNA enters the A site pairing codon with anticodon
- Elongation factors act as transporters to bring in the aminoacyl tRNA
- Peptide bond formation and covalent bond between tRNA and amino acid in P site is broken
- Uncharged tRNA at P site
- tRNA enters E site, two move to the P site a new one joins at A site (shifts by 3 bases)
- Aided by elongation factors
- Elongation steps are repeated
- Stop codon enters A site - release factors stimulate hydrolysis of the polypeptide from the tRNA
- Polypeptide released
What is the coding strand?
Has the same sequence as the RNA produced
What are UTRs?
Untranslated regiom - non-coding sequence
What is an ORF?
Open Reading Frame - part that is translated
Where can gene expression be controlled?
DNA (transcription initiation). RNA - how stable. Translation - translate or not. Protein- stability.
Is RNA more or less stable than DNA?
Less
What is the key to regulating transcription initation?
Regulating the interaction of RNA polymerase with the promoter region.
What are regulatory proteins?
Transcription factors. Regulate RNA polymerase.
What is positive regulation?
- RNA polymerase doesnt have strong affinity for its promoter
- Needs an activating transcription factor to attract it and turn transcription on
- Activator proteins need to be able to recognise specific DNA sequences
What is negative regulation?
- RNA polymerase is bound to the promoter and is transcribing
- Gene is expressed unless a repressor protein is present to turn it off
- Recognises specific sequences
How is control of gene expression in prokaryotes special?
Can change their gene expression to suit their environmental conditions
What do regulatory proteins in prokaryotes do?
Respond to changes in environment.
What are the 2 forms of regulatory proteins in prokaryotes?
One form can bind and regulate gene expression. One form doesn’t. Shape will change.
Where is the promoter region of regulatory regions?
Upstream. RNA polymerase and activating transcription factor proteins bind
What is the operator region?
Where repressor transcription factor proteins bind. Blocks RNA polymerase
What does the Lac Operon allow for?
Allows synthesis of lactose metabolising enzymes in E.coli when lactose is present as a carbon source.
What does B galactosidase do?
Metabolises lactose
What are the structural genes in the Lac Operon?
B galactosidase (lacZ). Permease (lacY). Transacetylase (lacA).
How is the Lac Operon regulated?
Through repressor protein (lacI)
What does the LacI gene do on the lac operon?
Produces I repressor protein - binds to operator - prevents transcription of lac operon
What happens if lactose is present?
LacI repressor binds to lactose. LacI changes shape and can’t bind
What is allosteric regulation?
Regulation of a protein by binding of an effector molecule at a site other than the enzymes active site or DNA binding domain. Causes a conformational change
What are the 2 domains of LacI?
One for DNA binding. One for lactose.
What is the Operator Constitutive?
LacI repressor would normally be recognising a sequence of DNA. Doesn’t repress
What happens to the lac operon if glucose is present?
Glucose is a better carbon source than lactose. If glucose is present the lac genes are not expression. Controlled by the CAP transcription factor.
What happens if glucose and lactose are present?
Glucose used first - lag phase (lac operon being translated) - then lactose is used
How is glucose levels monitored?
via cAMP - produced from ATP. cAMP catalysed by adenylate cyclase. Low glucose = cAMP levels increase
Describe what happens if glucose is present and the involvement of CAP
Glucose - cAMP low - no lactose - no lac mRNA. CAP doesn’t bind (no positive regulation)
Where does translation take place?
Cytoplasm.
What is the difference in translation in eukaryotes and prokaryotes?
Eukaryotes = has to move. Prokaryotes = can be translated as soon as its transcribed.
Where are ribosomes found?
Cytoplas. RER
Give an example of when gene expression goes wrong.
Drosphilia = legs instead of antennae. Gene which switches off legs in that section doesn’t seem to work
What are physical consequences due to?
Gene expression - regulatory system rather than the DNA itself
How is DNA packaged?
Highly ordered - compact. Difficult to transcribe. Different levels.
What is chromatin?
DNA and proteins. Condensed
What is heterochromatin?
Dense packagin. Little transcriptional activity.
What is Euchromatin?
More open, location of most protein coding genes.
What is postition effect variegation?
Evidence that chromatin packaging can influence gene expression.
What is meant by a dynamic structure?
Nucleosomes become more spaced out
What is in active gene?
Regularly spaced nucleosomes
What is an active gene?
Opening up of chromastin structure so there can be chromatin remodelling.
What does the opening up of the chromatin require?
Protein complexes that can perform chromatin remodelling
What does the opening up of the chromatin allow?
Access for RNA polymerase
What is the core promoter?
RNA polymerase assembles here.
Establishes start site for transcription initiation and includes the TATA box and initiation site
Promoter elements are upstream of the TATA box
What are basal promoter elements?
Set the basal level of transcription
What are the response elements?
Allow certain genes to respond to environmental stimuli
What are cell specific elements?
Located in promoters of genes expressed in only one type of tissue
Where can regulatory sites be found?
Way upstream or downstream
What can increase the level of transcrption?
Multiple proteins upstream or downstream to get RNA polymerase to bind. ‘Enhancer’ elements.
Describe the multiple binding sites?
Some responsible for opening chromatin. Help recruit RNA polymerase. Some cells type specific.
What are TERT promoter mutations?
The human TERT gene encodes for telomerase reverse transcriptase
Gene is normally transcribed at low levels in most adult differentiated cells
Common mutation in many cancers occurs within the TERT promoter and creates a new binding site for a transcription factor
Result is increased expression of TERT that contributes to cancer cell immortality
What is the structure of mRNA?
exons and introns
What happens to exons?
Join together to form the mature mRNA
What happens to the introns?
non-coding sequences that are removed from the primary transcript
Where does splicing occur?
In the nucleus at the spliceosome. Formed of RNA and protein.
What is Alternative Splicing?
Alllows a single gene to produce multiple protein produces. Increasing protein diversity
What is RNA localisation?
Where to gove
What can determine where splicing occurs?
Regulatory proteins
What is a missense mutation?
Mutation that results in an amino acid change but does not change the reading frame of the protein
What is a silent mutation?
A mutation that does not result in a change in amino acid
What is a frameshift mutation?
A mutation involving insertion or deletion of bases that is not a multiple of 3. This shifts the reading frame in all codons following the site of mutation
What is a nonsense mutation?
A mutation that changes a codon specifying an amino acid into a stop/termination codon
What are loss of function mutations?
Usually recessive and involve either complete loss or reduction in protein function
What are gain of function mutations?
Usually dominant and involve either an increase in protein function, acquisition of new function oor acquiring the ability to interfere with the function of the wild-type protein.
