Transcription and RNA Processing Flashcards
RNA features
-has uracil in place of thymine
-has a ribose sugar (it bears a hydroxyl (–OH) group on its 2’ carbon)
* tertiary structure
* may interact as functional units (quaternary
structure)
Why is RNA unstable and why use it?
The presence of the unique 2’OH group in ribose causes it to react intramolecularly with the 3’OH site resulting in phosphate bond breakage
-can form many tertiary structures allowing it to have
different conformations for different functions
-easier to control, easily degraded
What is transcription
synthesis of RNA from DNA templates
How does RNA ultimately lead to protein?
RNA is transcribed into mRNA (messenger RNA), which is translated into a protein
components for transcription
- DNAtemplate
- 4 ribonucleosidetriphosphates
(rNTPs)
* A, U, C, G
RNAn + rNTP RNAn+1 +PPi - DNAdependant RNA polymerase
RNA Polymerase Holoenzyme subunit functions
alpha -involved in the assembly of the tetrameric core (2 of these)
beta – contains the ribonucleoside
triphosphate (rNTP) binding site
beta prime- contains the DNA template binding region
ω : (omega)-helps to stabilize the tetrameric core
sigma-binds to RNA polymerase
tetrameric core and assists in the correct
initiation of transcription specifically at the
promoter region of the prokaryotic gene. Many types of sigma factors → allows for specificity
Rho-INdependent Termination
Poly A sequence is transcribed into a poly U
tail after the hairpin is transcribed.
* pauses poly
* Hairpin forms and destabilizes the DNA-RNA hybrid
* Assisted by the weak A-U base pairing
Rho-dependent Termination
Two sequence features:
1) DNA sequence of terminator site causes Polymerase to pause
2) DNA sequence upstream of terminator encodes a stretch of RNA that is C rich and devoid of secondary structure.
a) Called the rho utilization (rut) site.
b) Rho binds to rut site
* Rho moves along RNA towards paused Polymerase
* Rho factor has helicase activity
* Unwinds the RNA-DNA hybrid
* Brings transcription to an end
What are the roles of specific promotor sequences in eukaryotic transcription?
-special promoter sequences for genes transcribed by RNA poly I, II, or III
-accessory proteins recognize the specific promoters and recruit the appropriate poly to beign transcription
Transcription in Eukaryotes-Initiation
-assembly of transcription factors of Pol II (TFIID)
-TFIID complex contains TATA binding protein, assembles at TATA box followed by remaining TF’s and Pol II, causes DNA to unwind
-forms preinitaiton complex
Transcription in Eukaryotes-elongation
-poly moves along template strand leaving TF intact at promoter
-maintains rNTO transcription bubble
-hybrid bends at right angle
-positions -OH group at active site where nucelotides are added
-newly synthesized RNA separated from DNA and exits throguh cleft
Transcription in Eukaryotes-termination
-RNA Pol I:
requires a termination factor similar to rho factor in prokaryotes.
-RNA Pol II: transcription
continues past termination
sequence.
- RNA is cleaved at a
consensus sequence in the R
RNA Pol III: ends after transcribing a terminator sequence that produces a string of U’s that is downstream from a hairpin. Similar to rho-independent termination.
Cleaved RNA=2 RNAs: one
that will encode a protein and the
other with its 5’ trailing out of the
RNA polymerase.
* Rat1 (5’→ 3’ exonuclease) attaches
to 5’ end, degrades remaining RNA strand
* Transcription is stopped when
Rat1 reaches transcription
machinery
Transcription in Prokaryotes-Initiation
-sigma factor associates with core enzyme to form a holoenzyme which binds to -35 and -10 consensus sequences in promotor
-RNA poly positions over start site, and unwinds DNA to produce single-stranded template
-Initiation: RNA polymerase binds, unwinds and joins first 2 nucleotides (no primer)
Transcription in Prokaryotes-Elongation
-Elongation: when Sigma factor is released and RNA poly moves along the 3’ to 5’ DNA template strand. Nucleotides continuously added. RNA poly generates transcription bubble that moves with RNA poly
Transcription in Prokaryotes-Termination
-Termination: when RNA polymerase reaches “terminator” region of the gene.
* Occurs upstream of where the actual termination will take place.
* The newly-synthesized RNA together with the RNA polymerase are released.
* Bacterial cells possess two major types of terminators:
Rho-dependent (requires Rho factor) and Rho-
independent (aka intrinsic terminator)
exons
-protein coding segments
-vary in length and position on gene
What does RNA processing require in eukaryotes
pre-mRNA is produced from a template strand of a gene being transcribed, then is processed to make mature messenger RNA (mRNA) that can be translated into a protein
Three primary regions of a mature mRNA
- 5’ untranslated region (5’UTR): does not code for amino acids. Binds the
ribosomal complex. - Protein coding region: comprises the codons that specify the amino acids.
Begins with a start codon and ends with a stop codon. - 3’ UTR: does not code for amino acids. Affects the stability of the mRNA and
regulates its translation.
introns
-intervening (non-coding) segments
-can influence how genes make proteins or how non-coding RNA are expressed
-present on eukaryotic DNA, can vary in nucleotide length
-removed from pre-mRNA by action of a spliceosome
-has 3 conserved sequences:
1. 5’ splice sequences containing the junction sequences GU
2. 3’ splice sequence containing the junction seequence AG
3. intron branch point: a conserved “A” residue located upstream of 3’ splice site
addition of 5’ cap
facilitates binding of ribosome to 5’ end of mRNA, increases mRNA
-Addition of 7-Methyl Guanosine (7-
MG) Cap
-Linked to pre-mRNA by linkage between the 5’ P of the 7-MG and the 5’ P of the first ribonucleotide in the RNA (5’ to 5’phosphate
linkage)
3’ cleavage and addition of poly A tail
increases stability of mRNA, facilitates binding of ribosome to mRNA
-A’s added w/o template
-“polydenylation”
RNA splicing
removes noncoding introns from pre-mRNA, facilitates export of mRNA to cytoplasm, allows for multiple proteins to be produced
-removal of introns from pre-mRNA, must be precise to properly fuse the 3’ end of one exon to the 5’ end of the next exon
1. U1+U2 bind
2. U4, U5, U6 join complex. changes shape + proximity of U2+U6
3. U1 + U4 released
4. remaining spliceosome held by RNA base pairing
5. following cleavage, intron binds (lariat) and exons join (w p-bonds)
6. introns released
spliceosome
-RNA/protein structure
-has 5 snRNAs (small nuclear RNAs) that associate with 300 small proteins to form snRNP (small nuclear ribonucleoproteins
spliceosome assembly
-snRNA of spliceosome form complimentary base pairing with pre-mRNA
-cataylytic steps carried out y snRNAs of the spliceosome
-addition of U4, U5 and U6 join the spliceosome
-U4, U5, U6 cause shape change
-U1 and U4 dissociate
-base pairing occurs between mRNA and snRNA sequences that hold the splcieosome together
-2 transesterification rxn by U6, joins 2 exons together and releases the lariat
alternative processing pathways
alternative splicing (introns)
-pre-mRNA processed to produce diff types of mRNA
-produces diff proteins from same DNA sequence
multiple 3’ cleavage site (exons):
-can generate longer/shorter exon (on last exon)
-may or may not produce diff protein
RNA editing
changes gene content via
-editing mRNA by endogenous guide RNAs
-inserting/deleting uridine monophosphate residues
-changes structure of individual bases (tRNAs, mRNAs)
what are endogenous guide RNAs and nucleotide addition in RNA processing
-gRNAs made in cell direct insertion of uridine bases into mRNA by repair polymerase
-permanently modifies the mRNA by making new codons that specify new amino acids
tRNA modifications
-anticodon of tRNA base pairs with mRNA codon
-amino acid covalently attached to tRNA 3’ end
-contain modified ribonucleotides through modifying enzymes
(extra 3’ and 5’ sequences removed, introns removed)
snRNA
small nuclear RNA
-acts in complex with proteins
-plays role in post-transcriptional processing of RNA (splcing)
snoRNA
small nucleolar RNA
-act in complex with proteins
-in eukaryotes, guide enzymatic chemical mods of ribosomal RNA in nucleolus
SiRNA and miRNA
small interfering RNA and micro RNAs
-in eukaryote as double-stranded RNA
-act as short, single stranded RNAs, binds to complementary sequences in mRNA
-produced by mRNA cleavage, RNA transposons + RNA viruses
-regulates gene expression
crisprRNA
in prokaryotes
-encoded by DNA sequences in rokaryotes genomes
-works with Cas9 nuclease to cleavage foreign DNA
long-non coding RNA
in eukaryotic cells
-regulate gene expression at transcription and translation level by binding mRNA or isolating micro-RNAs that control gene expression
