24 transcription Flashcards
Where does DNA reside and where does protein synthesis take place?
DNA reside in nucleus
protein synthesis in ribosome in cytoplasm
What are the 2 hypothesis on how DNA is moved from the nucleus to the cytoplasm?
- rRNA in ribosomes carries message from gene; each ribosome is specific for one kind of protein
- ribosomes translate an mRNA specified by gene; ribosomes can make many different proteins
How did they discovery that mRNA that carries info from DNA to ribosome?
- grow bacteria on medium containing heavy isotopes (bacterial ribosomes are labeled with heavy atoms)
- infect bacteria with phage which destroy bacterial DNA. switch to light medium = end up with bacterial ribosome with viral DNA
- growth medium contains radioactive uracil to label newly synthesized viral RNA
- bacteria lysed and centrifuged on a CsCl gradient
finding
* only heavy (bacterial) ribosomes were detected, so phages did not direct the synthesis of new ribosomes for protein synthesis
* radiolabeled RNA was found to be associated with old bacterial ribosomes
* the radiolabeled RNA on ribosomes hybridized to phage DNA. so the newly synthesized phage RNA are complementary to phage DNA - mRNA
so what was found in the discovery of mRNA?
- expression of viral genes is associated with the formation of viral-specific RNA (mRNA)
- mRNA form complex with ribosome
- mRNA carry information from DNA to the ribosome for protein synthesis
- ribosomes are passive sites of synthesis
Within each gene, which strand of DNA is transcribed?
the template strand (3’ to 5’)
* the non template strand (5’ to 3’) is the copy that we want
What is the function of rRNA and where can it be found?
structural and functional components of the ribosome
found in cytoplasm
What is the function of mRNA and where can it be found?
carries genetic code for proteins
found in nucleus and cytoplasm
What is the function of tRNA and where can it be found?
helps incorporate AA into pp chain
found in cytoplasm
what can both prokaryotic and eukaryotic ribosomes be broken down into?
2 subunits (large and small)
how are ribosomes made?
rRNA transcribed in nucleus then leaves to cytoplasm and assembles as ribosomes
What are required for transcription?
- DNA template
- ribonucleotides
- RNA polymerases: enzymes that catalyse the synthesis of RNA
- accessory proteins: e.g. sigma factors, general transcription factors
In what direction is the template DNA copied in transcription?
is copied from 3’ to 5’
where does RNA polymerase add ribonucleotides?
adds ribonucleotides to 3’-OH ends, following watson-crick base pairing
How is the RNA strand compared to DNA template?
is complementary and antiparallel to the DNA template
What base change is used in RNA compared to DNA?
U instead of T
What is RNA polymerase core made up of in bacteria?
αββ’ω
What is the difference between RNA polymerase I, II, III in eukaryotes?
- RNA polymerase I transcribes gene coding for large rRNA
- RNA polymerase II transcribes protein coding genes to give mRNA
- RNA polymerase III transcribes small RNAs e.g. tRNA etc
What does a transcription unit include?
- promoter
- an RNA-coding region
- terminator
can different genes be transcribed from one or 2 DNA strands?
yes
different genes may be transcribed from one or the other of the 2 DNA strands
both can be used
What is the structure of a typical bacterial promoter?
- transcription start site (at +1)
- consensus sequences at around -35 and -10 positions relative to the start site, they are specific sequences that are recognized by sigma factors
- different sigma factors recognize different promoter sequences
- sigma factors help RNA polymerase to recognize and bind to the promotor
what are primary sigma factors?
Generally involved in the transcription of essential genes for basic bacterial functions.
What are specialized sigma factors?
different sigma factors recognize differnt promotor sequences = ask diff genes to be transcribed
What does the bacterial core RNA polymerase need to bind to the promotor?
sigma factor
What is UP?
upstream promotor
Why do some promoters of bacterial genes contain UP (upstream promotor) element?
located upstream of the core promoter. This region stabilizing RNA polymerase binding to the promoter, and increasing the transcription rate.
What is the structure of RNA polymerase II promotors in eukaryotes?
- BRE: TFIIB recognition element
- TATA: TATA box (the consensus sequences)
- INR: initiator element
- DPE: downstream promoter element
What are the 3 steps of transcription?
- initiation
- elongation
- termination
What happens in initiation of transcription?
- RNA polymerase binds to promoter DNA sequences
- RNA polymerase seperates the DNA strand to create a transcription bubble (promotor opening)
- ribonucleoside triphosphates (rNTP) are added
- Polymerase moves past the promotor and becomes stably bound to DNA (promoter clearance)
Promoter clearance occurs when RNA polymerase moves beyond the promoter region and continues elongating the RNA transcript.
What happens in elongation of transcription?
- RNA polymerase moves along template strand and elongate the RNA transcript
- RNA polymerase unwinds duplex ahead of it to expose single strand template. DNA strands behind RNA polymerase pairs again to reform DNA duplex
- the growing RNA trasncript is extruded through the RNA polymerase
What happens in termination of transcription?
RNA polymerase dissociates from DNA template when it encounters a terminator sequence
Can many RNA polymerase act at once?
yes
many places where transcription is happening and they can start at different times on the same strand of DNA
In eukaryotes, what does transcription produce?
pre-mRNA
How can pre-mRNA be further processed in eukaryotes
- addition of 5’ cap: a nucleotide with 7-methylguanine; 5’-5’ bond is attached to the 5’ end of the RNA
-
addition of poly(A) tail: 50-250 adenine nucleotides are added to the 3’ end of the mRNA
* RNA splicing: introns removed exons spliced togehter
What are 3 function of 5’ capping in eukaryotes?
- recognized by the cap binding complex for nuclear transport (leave nucleus to cytoplasm)
- prevents degradation by 5’ exonucleases (increase the half life of mRNA for export and translation which take significant time)
- aids translation initiation
What are 3 functions of poly(A) tail?
- protects the mRNA molecule from enzymatic degradation in the cytoplasm (5’ capping protects 5’, also need to protect the rest)
- aids transcriptional termination
- aids translation initiation
How is poly(A) tail added?
through cleavage and polyadenylation
What is polyadenylation?
A process that adds a poly(A) tail to the 3’ end of pre-mRNA, crucial for RNA stability and translation.
What are the components of polyadenylation complex?
Cleavage and Polyadenylation Specificity Factors (CPSF):
* Bind to a poly(A) signal sequence (typically AAUAAA).
* Interact with other factors to stabilize the assembly.
CstF (Cleavage Stimulation Factor):
* Binds to a downstream G/U signal.
* Forms a complex with CPSF.
CFI and CFII:
* Assist in stabilizing the CPSF-CstF assembly.
* Help form a loop in the RNA.
Poly(A) Polymerase (PAP):
* Binds to the poly(A) site.
* Stimulates the addition of the poly(A) tail.
What is the process of polyadenylation
- CPSF and CstF bind to the pre-mRNA at specific sequences.
- CFI and CFII stabilize the complex, facilitating the formation of a loop.
- PAP adds adenine residues (poly(A) tail) to the 3’ end of the mRNA.
What is splicing in pre mRNA?
removing the introns
How do spliceosome remove introns from the pre-mRNA?
spliceosome recognize the splicing site and form a complex to generate the cleavage and remove the intron
What sequence do introns typically have at the 5’ end and 3’ end
intron typically have a GU nucleotide sequence at the 5’ end splice site and an AG at the 3’ end splice site
what are spliceosomes composed of?
- 5 small nuclear RNAs (present only in eukaryote)
- U1, U2, U4, U5, U6
- and a number of associated protein factors
How do bacterial and eukaryotic genes compare in terms of introns and exons?
Bacterial genes - do not contain introns
eukaryotic genes - contain introns and exons
What is alternative splicing?
the re-combination of different exons to generate genetic diversity
diff protein from one mRNA
use less DNA and mRNA to make more protein
what are UTRs?
untranslated regions, sectinos of mRNA that dont get translated
Where is 5’ and 3’ UTR relative to coding sequence?
5’ = upstream from the coding sequence
3’ = immediately after the termination codon in coding sequence
What is the role of 5’ and 3’ UTR?
5’ = it is recognized and bound by the ribosome to initiate translation
3’ = contains binding sites for microRNAs
Can microRNAs be found in prokaryotes and eukaryotes?
only in eukaryote
Are microRNAs transcribed?
yes usually by RNA polymerase II, some by RNA polymerase III
What is the function of microRNAs?
decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcripts
by generating a mature small piece of microRNA -> recognize mRNA transcription at 3’ UTR region and ask them to be degraded
Regulate level of mRNA in cell
What can regulate the level of mRNA in cell?
micro RNA
If micro has complementary base w mRNA within a seed region
Then that particular mRNA can be targetted
One miRNA can target many mRNA
Many possible matching of diff RNA
What are 3 sources of microRNA?
- Polycistronic: Some microRNAs are found within the coding regions of genes/Encoded Within Genes
- Intergenic: MicroRNAs can also be located in intergenic regions/between genes.
- Intronic/Exonic: Some microRNAs are derived from introns or exons of pre-mRNA.
How can micro RNA regulate mRNA in 3 ways
- Translation: Inhibition of protein synthesis.
- Degradation: Breakdown of target mRNA.
- Deadenylation: Removal of the poly(A) tail, affecting stability.
What are processing bodies (P-bodies)?
cytoplasmic structures involved in the regulation of mRNA. Where mRNAs that are no longer needed are degraded w many enzymes
What are 2 similarities of replication vs transcription?
- both require a template
- strand extend from 5’ to 3’, antiparallel to template
What are the differences between replication and transcription?
number of strands
whats copied
how many copies
What happens to the newly synthesized strand
nucleotide usage
primer?
