Theme 2B Flashcards
Transcriptional and Post-Transcriptional Gene Regulation
Transcriptional regulation of gene expression
- Initiation
- Elongation
- Termination
Differential expression of genome
- when not all genes are being turned on
- gives rise to different cell types and tissues
Organism’s phenotype is dependant on
cell number, type, and function
Every cell in an organism has an ____________
identical genome (DNA sequence)
Turning on a gene means going from ______ to __________________________
DNA; transcription
Structure and function of a gene
gene includes a promoter and transcriptional unit
Promoter
DNA sequence (includes TATA box) that specifies where transcription begins on chromosome
- signal or landing spot for enzymes so location of specific gene can be identified
- located immediately upstream or 5’ of the transcriptional starting point of the non-template or coding DNA strand
- bound and recognized by transcriptional machinery
Transcriptional machinery
initiate transcription
- RNA polymerase and transcription factors
Transcriptional unit
part of the gene that is copied into RNA
RNA polymerase: enzymes of transcription
synthesize RNA transcript in a 5’-3’ direction while reading DNA template in the 3’-5’ direction
- does not need primer for initiation of RNA synthesis
- unwinds and rewinds DNA helix during RNA synthesis
3 types of RNA polymerase
RNA polymerase I
RNA polymerase II
RNA polymerase III
RNA pol I
transcribes rRNA
RNA pol II
transcribes mRNA
RNA poly III
transcribes tRNA
Step 1: Transcriptional Initiation
transcriptional initiation is mediated by direct interaction of DNA-binding proteins to specific regulatory sequences of the gene (rate determining step)
2 types of processes:
- general transcriptional factors bind to promoter and recruite RNA poly II resulting in LOW BASAL LEVEL of transcription (gene expression = low)
- transcriptional activator proteins bind to enhancer regions distant from promoter to cause DNA looping bringing mediator and RNA polymerase to promoter resulting in HIGH LEVEL of transcription
Step 2: Transcriptional Elongation
RNA pol moves along template DNA (3’ to 5’)
DNA is unwound in front of moving RNA poly and reannealed behind in the transcription bubble
Ribonucleotides are added to the 3’ end of the RNA transcription (synthesis continues 5’-3’)
growing RNA transcript is displaced from DNA template strand to allow reannealing back into double stranded DNA
Step 3: Transcriptional Termination
5- sequence in DNA template causes termination after transcribed into RNA
- Rho-independant termination
- Rho-dependant termination
- Cleavage & polyadenylation specifc factor
- Rho-independant termination
prokaryotes
terminator sequence in mRNA base pairs with itself to form G-C hairpin and causes RNA polymerase to stall and dissociate
- Rho-dependant termination
prokaryotes
terminator sequence in mRNA is recognized and bound by the Rho helicase which unwinds the RNA from template DNA and RNA polymerase
- Cleavage & polyadenylation specific factor
eukaryotes
poly-A sequence in mRNA signals the CPSF to cleave the completed mRNA transcript thereby separating it from RNA polymerase
When is the RNA molecule the longest during transcription
end of transcription as it reads more
When is the RNA molecule the shortest during transcription
start of transcription as it reads more as you go
In transcription, RNA molecules are _____________
single/double strands?
single strands
transcription occurs at _______ in the genome
selected locations
In transcription, synthesis of RNA occurs in __________ and copies vary throughout genome
multiple copies
In transcription, RNA poly does not need a _________ for initiation
primer
In transcription, RNA product does not remain ________ to the template DNA
base-paired
In transcription, synthesis of RNA occurs in the _________ direction
5’-3’
In DNA replication, DNA molecules are
single/double stranded?
double stranded
In DNA replication, replication occurs for the ___________
entire genome
In DNA replication, genome is only replicated _____________
once/cell cycle
In DNA replication, DNA poly requires __________ for initiation
primer
In DNA replication, daughter strand remains ____________ with parental template strand
base-paired
In DNA replication, synthesis of new DNA strand occurs in the ______ direction
5’-3’
Posttranscriptional regulation of gene expression has 3 steps:
- 5’ capping
- 3’ polyadenylation
- splicing
The ends of prokaryotic and eukaryotic mRNAs are __________
not translated
Both 5’-UTRs and 3’-UTRs regulate
mRNA stability and translational efficiency
5’-UTRs contain
ribosome binding stie (RBS) or Shine Dalgarno sequence in prokaryotes
&
Kozak box sequences in eukaryotes
that function in translational initiation
Open reading frame (ORF)
region of mRNA that is translated and includes the start and stop codons at the borders
The newly transcribed mRNA (pre-mRNA) undergoes processing in the ________ to produce ___________
nucleus ; mature translatable mRNA
- 5’CAP
modified guanosine triphosphate is added to the 5’ end of the mRNA and acts as a ribosome binding site (attracts r to 5’) and protects mRNA from degradation
Poly(A) tail
long string of adenine nucleotides added to the 3’ end of the mRNA by poly-A polymerase to protect the mRNA from being degraded & increase translational efficiency
Introns are removed/spliced during ___________ to ____________b/c
pre-mRNA processing; produce translatable mRNA because they don’t contain codon
If you remove poly (A), what enzyme degrades?
5’-3’ exoribonuclease
The longer the poly-A, the more
stable
Posttranscriptional processing from pre-mRNA to mRNA
newly-transcribed precursor mRNA (pre-mRNA) needs to be converted to a translatable mRNA because it cannot be yet translated into a protein
- addition of 5’-CAP and poly-A tail
- pre-mRNA has a mix of alternating coding segments and UTRs (exons) and non-coding segments (introns)
- removal of introns by splicing to generate the open reading frame consisting of a continuous stretch of codons & URTs
- mRNA is exported from nucleus into the cytoplasm to associate with ribosomes
Why is removal of introns by splicing important?
if they are not removed, you won’t get functional protein
Exons
coding segments and UTR
Introns
non-coding segments
mRNA splicing
removal of introns from pre-mRNA and joining of exons to make mature mRNA
spliceosome
- carries out splicing
- made out of snRNA and splicing proteins
- made up of five non-coding RNAs (snRNA) complexed to several proteins (small ribonucleoprotein particles/ snRNPs)
Process of Splicing in 4 steps
- Spliceosome binds to intron-exon junctions
- Loops introns out of the pre-mRNA (lariat structure) bringing exons closer together
- Clip the intron at each exon boundary releasing the lariat structure
- Join adjacent exons together
Alternative splicing
splicing can occur in different combinations to generate two or more different mRNAs from a gene, and several related protein products (isoforms)
- different isoforms are made in diff tissues from the same gene, producing tissue-specific phenotypes
- increases the number and variety of proteins that can be encoded by a genome
Posttranscriptional regulation by RNA interference
found in all eukaryotes
microRNA transcribed by RNA Pol II; small interfering RNA also transcribed (siRNAs)
- miRNA/siRNA precursors are cleaved to 21-23 bp double-stranded RNAs by the Dicer Rnase
- these double stranded RNAs are substrates of RISC
- likely evolved as an antiviral mechan9ism to destroy viral mRNA
RISC
RNA induced-silencing complex
- unwinds one of the RNA strands which attracts binding of complementary mRNA
(Posttranscriptional regulation by RNA interference)
Binding of the mRNA to RISC interferes with ____________ or induces ___________
translation initiation; mRNA degradation (represses gene expression)
Transcriptional regulation
control of mRNA synthesisT
Transcription rate depends on the
speed of transcriptional initiation (promoter strength)
Posttranscriptional regulation
processing of mRNA which affect- usually improves - its stability & translational efficiency
Stability of mRNA depends on the presence of ____________ & _______________
5’-CAP & length of poly-A tail
Expression level of a specific gene depends on the ___________, ____________, and it’s eventual _______
abundance of mRNA, nucleotide sequence, eventual translation
Abundance of RNA depends on
rate of synthesis (transcription) and degradation of mRNA (posttranscriptional)
More RNA = More
Protein
- depends on RNA stability
If the enhancer is deleted…
Level of gene regulation affected=
Gene expression increases/decreases
Molecular process affected =
- transcription
- decreases
- RNA polymerase cannot initiate transcription (gene expression decreases)
If polyadenylation of mRNA is increased…
Level of gene regulation =
Gene expression increases/decreases
Molecular process affected =
- post-transcription
- increased
- enhanced mRNA stability & translation
If 5’-CAP is removed…
Level of gene regulation =
Gene expression increases/decreases
Molecular process affected =
- post-transcription
- decreases
- mRNA degradation accelerated, less efficiency in translation initiation
If TATA box is deleted….
Level of gene regulation =
Gene expression increases/decreases
Molecular process affected =
- transcription
- decreases
- formation of transcriptional initiation complex is hindered
If siRNA synthesis is inhibited….
Level of gene regulation =
Gene expression increases/decreases
Molecular process affected =
- post-transcription
- increases
- less mRNA degradation
Review slide 20
