Mod 4- From DNA To RNA Flashcards
What is a gene?
A genetic unit containing information to make a functional product (RNA and/or Protein)
Genes can carry information for structural, temporal, positional, and inducible functions.
What information does a gene contain?
- Structural - coding DNA
- Temporal (developmental= when gene switches on/off)
- Positional (tissue/cell specific= when it switches on/off)
- Inducible (nutrients, stress, hormones= switching genes on/off due to environment)
These categories help define the role and regulation of genes in different contexts.
What is a cistron?
A segment of DNA corresponding to one polypeptide (stop and start signals)
What is the role of a promoter in gene transcription?
Defines transcription start site and its direction (for RNA polymerase)
Promoters are essential for the binding of RNA polymerase
What is the difference between sense and antisense strands?
- Sense (coding) strand: 5’ - 3’ sequence matches RNA product
- Antisense (template) strand: 3’ - 5’ sequence is complementary to RNA product
Understanding these strands is crucial for studying transcription.
What are the key features of eukaryotic Class II genes?
- Enhancer = sometimes distal from promoter, contains transcription factor binding sites (works in a 5’ direction)
- Promoter
- Exons = in mature transcript
- Introns = in primary transcript but removed from mature transcript
- 5’-UTR
- 3’-UTR
UTR = untranslated region
Eukaryotic genes are more complex than prokaryotic genes and include regulatory elements.
What is a polycistronic mRNA?
An mRNA that contains multiple cistrons, allowing for the expression of multiple proteins
This is a common feature in prokaryotic gene organization.
Fill in the blank: Eukaryotic genes can be quite large, approximately _______ kB.
50
Most of this size is due to non-coding regions.
What is the general mechanism of transcription?
- Coding strand (sense): 5’ - 3’ = same sequence as RNA product = leading strand
- Template strand (antisense): 3’ - 5’ = complementary to RNA product = lagging strand
What is the function of RNA polymerase?
Binds to DNA, melts double strand, and polymerizes RNA in the 5’ to 3’ direction
RNA produced is copy of the coding strand and complementary to template strand
What is the significance of the TATAAATG sequence?
It is part of the promoter region that helps RNA polymerase bind to DNA
This sequence is crucial for the initiation of transcription.
True or False: All genes contain a regulatory element.
True
This is the promoter/ enhancer
What distinguishes prokaryotic gene organization from eukaryotic gene organization?
- Cistrons vs. introns/exons
- Lack of complex regulatory elements in prokaryotes
Understanding these differences is important for molecular biology.
What are the untranslated regions (UTR) in eukaryotic genes?
- 5’-UTR
- 3’-UTR
UTRs are crucial for the regulation of translation and stability of mRNA.
How many RNA polymerases are present in prokaryotes and eukaryotes?
Prokaryotes have one RNA polymerase - multi-protein complex that does all of the functions and makes all types of RNA
Eukaryotes have three
Eukaryotic RNA polymerases include Pol I, Pol II, and Pol III
What are the roles of the components in the prokaryotic RNA polymerase holoenzyme?
RNA polymerase holoenzyme is composed of 6 subunits
sigma (σ)
- promoter recognition
- recognises and binds to promoter region of a gene to unwind the DNA slightly allowing transcription to begin (‘guiding’ factor)
- recognises specific DNA sequences (promoter) and gives direction
2 x alpha (α)
- assembly, activation
- smaller
- activates the polymerase by assembling the core of the enzyme
2 x beta (β + β’)
- catalysis, termination
- opens and polymerises DNA to make RNA copy
- large
- builds the RNA chain
- holds the DNA template in place within the active site and binds incoming RNA nucleotides
- signals end of transcription
Omega (ω)
- assembly, folding, required for some genes
- doesn’t do much in terms of function
- stabilises structure of the polymerase and protects it from denaturing
What is TFIID composed of?
TATA binding protein (TBP) + TBP-Associated Factors (TAFs)
TBP is A-T rich
TFIID is involved in the assembly of the RNA polymerase II initiation complex
It recognises the TATA sequence on DNA
What is the function of TFIIA?
Helps TFIID to bind to DNA
It facilitates the interaction of TBP with DNA
What does TFIIB do in the transcription initiation process?
An accessory factor
This is a single polypeptide
Sets distance from TATA element (where core promoter sequence is) to start site of transcription
What is the structure of the TBP-TFIIB-DNA complex?
Saddle structure
What is the role of the Upstream Binding Factor (UBF) in RNA Polymerase I transcription?
Binds to UCE and core element in DNA (loops DNA together)
Fill in the blank: RNA polymerase III is recruited and initiates transcription after the recruitment of _______.
TFIIIB
What is the function of TFIIIC in RNA polymerase III transcription?
Binds to B box and is recruited upstream of start site
Once TFIIIB is recruited, TFIIIC is dispensable
True or False: Eukaryotic RNA polymerases are specialized and gene-type specific.
True
What are the key differences in transcriptional apparatus between prokaryotes and eukaryotes?
Complexity and organization; prokaryotes have one polymerase, eukaryotes have three
Eukaryotic polymerases use general transcription factors
Identify the general transcription factors associated with eukaryotic RNA polymerases.
- TFIIA
- TFIIB
- TFIID
- TFIIE
- TFIIF
- TFIIH
- Others (TFIIJ, etc.)
What is the significance of the TATA box in transcription?
Recognized by TBP, crucial for the assembly of the transcription initiation complex
What is the function of the RNA polymerase II?
Has 10 subunits IN EUKARYOTES and has 6 subunits IN PROKARYOTES
TF2E,F,H,J,K
- Facilitate interaction with D-A-B complex and recruitment of polymerase II
- Blocks non-specific binding of polymerase II to DNA
- Promoter clearance = opens DNA and reads and makes RNA
- Helicase = mostly done by TFIIE
- Processivity and elongation = checks for errors, mostly done by TF2H
- Transcription-coupled DNA repair = done by TF2H
What is an operon?
A cluster of genes transcribed by the same promoter that gives rise to a polycistronic mRNA
Operons usually consist of genes that are related in function.
What does the lac operon do?
Hydrolyses lactose to galactose and glucose
What occurs when there is no lactose present in the lac operon?
The lac operon is repressed as the repressor binds to the operator, preventing RNA polymerase from clearing the promoter.
What role does allolactose play in the lac operon?
Allolactose binds to the repressor, causing it to dissociate from the operator, thus inducing transcription.
True or False: Allolactose is considered an inducer of the lac operon.
True
Gene only works if lactose is present
It is under negative feedback control ( basically self-regulating)
What are cis-acting elements?
DNA sequences that regulate only the genes to which they are directly joined.
These are dominant if mutated
‘Cis’ = next to/ alongside/ adjacent to
What are trans-acting factors?
Regulatory proteins that can regulate genes anywhere in the genome.
Mostly protein transcription factors
Recessive (mostly - phenotype can be rescued)
Define cis-acting mutations.
Mutations in regulatory elements that cannot be complemented and affect only the operon they are part of.
Define trans-acting mutations.
Mutations that can be complemented and may affect many genes.
What is catabolite repression?
The phenomenon where several operons are repressed by glucose and activate only when glucose is exhausted.
What is the role of CRP in the lac operon?
RNA polymerase requires CRP
CRP (Catabolite Repressor Protein) binds to cAMP, permitting DNA binding
High glucose = low cAMP so lac operon is off even if lactose is available
Low glucose = high cAMP so lac operon is on IF lactose is present
Glucose preferentially used as a carbon source
What happens to cAMP levels when glucose is high?
cAMP levels are low, causing the lac operon to be off, even if lactose is available.
Only produced when there is no glucose
cAMP = signalling molecule
What does the trp operon do?
Contains genes for the synthesis of the amino acid tryptophan.
What is the trp repressor?
The trp repressor binds to the operator in the presence of tryptophan and prevents transcription
Compare the lac and trp operons regarding their inducers and repressors.
Lac operon is induced by lactose (substrate), while trp operon is repressed by tryptophan (biosynthetic product).
Fill in the blank: Products of biosynthetic operons ______ their operons.
[repress]
Fill in the blank: Substrates for metabolism ______ their operons.
[induce]
What interactions are emphasized in the mechanism of regulation by operons?
- DNA-protein interactions
- Protein-protein interactions
- Protein-small molecule interactions
- Conformational change
- Inducers/co-repressors
What is a leader/ spacer?
Section of DNA which is non-translated
Transcribed into mRNA for regulating gene expression
NOT translated into protein
What is the organisation of a prokaryotic gene?
Start (AUG), leader, coding region (cistron), spacer (UGA) terminator
What happens in transcription initiation by prokaryotic RNA polymerase?
The sigma factor is released and the core enzyme (aabb’) is competent for elongation
Polymerase starts making RNA whilst still sat at the promoter = promoter clearance (to error check)
What are the 3 RNA Polymerases in eukaryotes?
Pol I - ribosomal RNA genes (rRNA)
Pol II - protein coding genes (mRNA) and small nuclear RNA (snRNA)
Pol III - transfer RNA (tRNA) and rRNA and snRNA
What do genes which make different types of RNA have?
- different architecture
- use different RNA polymerases (recognises different promoter sequences)
- each type of RNA polymerase needs a distinct set of accessory factors (in eukaryotes)
What is SL1 composed of in RNA polymerase I?
TBP (TATA binding protein) + pol I- specific TAFs (TBP-associated factors)
TBP & the TAF bind the promoter
What is the role of SL1?
Selectivity factor 1
This is specific to the species
Recognises promoter of the rRNA genes and initiates transcription when RNA Polymerase I binds to the complex
Explain the mechanism of RNA polymerase III
- Promoter is within the coding region
- TF3C (found to the right) binds to the B box and recruits TF3B
- TF3B (TBP sits at the centre of this) is recruited upstream of the start site
- TBP is non-contacting DNA acting as a saddle
- TF3B = TBP + 2 pol III- specific TAFs
- Once TF3B is recruited, TF3C is dispensable (not needed as transcription has started so gets displaced)
- RNA polymerase III is recruited and initiates transcription
What are other RNA polymerase III genes?
- 5S rRNA genes are similar to tRNA genes (has promoter sequence after the transcription start site) but use an extra factor TF3A to recruit TF3C
- snRNA genes have a similar structure to pol II genes = have an upstream promoter and TATA box
What are lacZ, lacY and lacA?
All code for a single RNA and each bit of this RNA codes for a separate polypeptide
lacZ - b-galactosidase = hydrolyses lactose (a disaccharide) to galactose and glucose (monosaccharides of lactose)
lacY - Permease = facilitates diffusion of lactose
lacA - Acetylase
What are cis-acting sequences?
Directly regulate the gene i.e. promoters
These are part of the gene
These are binding sites for trans-acting factors
