Module 8.1 Transcription and RNA processing Flashcards
RNA abundance by mass
- rRNA (80-90%)
- tRNA (10-15%)
- mRNA (3-5%)
RNA by number of molecules
- tRNA
- rRNA
- other RNAs
Gene expression regulation
factors (5)
- Which: gene function
- Where: cell and tissue types
- When: developmental stages
- Quantity: expression level
- Dynamic: external signals
Gene expression regulation
housekeeping genes
- common to all cells
- usually expressed universally in all the cells
- genes coding for structural proteins of chromosomes
- many proteins that form cytoskeleton eg. Actin
Gene expression regulation
Cell and tissue type
- some RNAs and proteins only expressed in specialized cells
- Hemoglobin expressed specifically in red blood cells where it carries oxygen.
- tyrosine aminotransferase breaks down tyrosine in food expressed in liver but not in most other tissues
Gene expression regulation
Developmental stages
- in early vertebrate development, Hox gene expressed at different times and locations
- controls formation of body structures along anterior-posterior axis
Gene expression regulation
Expression level
- level of RNA expression in different human cell lines of almost every gene found to vary from one cell type to another
- genes expressed in all cell types usually vary in levels of expression in different cell types
Gene expression regulation
External signals
- each cell is capable of altering pattern of gene expression in response to extracellular cues
- starvation or intense exercise: Glucocorticoid released in body signals liver to increase production of sets of proteins involved in generating energy from amino acids and other small molecules
- When hormone no longer present, protein production drops to normal unstimulated levels in liver cells
RNA polymerase
bacterial
- multi subunit complex
- subunit sigma factor is largely responsible for reading signals in promoter sequence that tells it where to begin transcription
RNA transcription
bacterial promoter
- special sequence on DNA double helix that indicates starting position for RNA synthesis
- heterogenous
- two hexamers at -35 and -10 positions (relative to transcription start position) that are relatively conserved characteristic sequences recognized by sigma factor
- nucleotide sequence between -35 and -10 hexamers differ among all promoter sequences
- promoter sequence -> promoter strength
- asymmetric - can only bind in one orientation
promoter strength
- number of initiation events by promoter per unit of time measured
- Promoters for genes of abundant proteins are much stronger than genes for rare proteins
Prokaryotic Transcription
process (7)
- RNA polymerase binds weakly to DNA and slides rapidly along DNA molecule until sigma factor recognizes promoter region
- polymerase binds tightly to promoter sequence
- Polymerase opens double helix to expose short stretch of nucleotides and use one of the two strands of DNA as template for RNA synthesis
- sigma factor dissociates and polymerase moves rapidly to elongate RNA chain until enzyme encounters terminator
- polymerase transcribes terminator sequence, hairpin structure forms and helps release RNA transcript
- polymerase dissociates from DNA
- polymerase reassociates with free sigma factor and searches for new promoter
Transcription regulator
- regulates transcription initiation which respond to extracellular signals
- Positions, identity, and arrangement of cis -regulatory elements determine time and place each gene is transcribed
transcriptional repressor protein
- transcription regulator that turns genes off
- genes that encode them continuously transcribed at low levels = fast response
transcriptional activator protein
prokaryotes
- transcription regulator that turns genes on
- poorly functioning promoters made fully functional by activator proteins that bind to nearby cis regulatory sequences and contact RNA polymerase to help it initiate transcription
- binding of activator to DNA often controlled by introduction of a signaling molecule like a metabolite or other small molecule
Prokaryotic transcription
terminator signal
- stretch of AT bases preceded by symmetric DNA sequence for most bacterial genes
- when transcribed into RNA folds into hairpin structure
transcription activator proteins
eukaryotes
- determines transcription rate and pattern
- can sometimes act from several thousand nucleotides away from promoter region
- help RNA polymerase, general transcription factors and mediators to assemble at promoter region
eukaryotic transcription
5’ capping
process
- phosphatase removes one phosphate from 5’ end of new RNA
- guanyl transferase adds guanosine monophosphate in reverse linkage to 5’ end
- methyl transferase, adds a methyl group to guanosine
- Cap binds to a protein complex called CBP or Cap binding complex, which helps the RNA to be properly processed and exported.
RNA splicing
- Both intron and exon sequences are transcribed into RNA
- The intron sequences are removed
- The exon sequences are joined into a continuous coding sequence
- machinery that catalyzes pre MRNA splicing involves multiple RNA molecules and hundreds of proteins.
- complexity needed to ensure splicing is highly accurate yet sufficiently flexible to deal with enormous variety of introns
- cell can easily regulate pattern of RNA splicing so that different forms of protein can be produced at different times in different tissues
RNA splicing
process
- specific adenine nucleotide in intron region attacks 5’ splice site and cuts sugar phosphate backbone of RNA
- cut 5’ end of intron becomes covalently linked to adenine nucleotide -> loop in RNA molecule
- released free 3’ hydroxy end of Exon sequence then reacts with start of next exon sequence, joining exons
- intron is released and degraded
Alternative splicing
- Exons can be spliced in a variety of different ways to produce different mRNAs and proteins from the same gene
mRNA 3’ end polyadenylation
process
- CSTF and CPSF proteins travel with RNA polymerase and bind to 3’ end processing sequence on RNA molecule as it emerges from RNA polymerase
- CPSF =AAUAAA hexamer
- CSTF = GU-rich region
- third factor = 3’ CA sequence - Additional proteins assemble for 3’ end modification
- RNA cleaved from cleavage site
- PolyA polymerase (PAP) adds ~200 A nucleotides one at a time to cleaved 3’ end
Types of RNA
Messenger RNA (mRNA)
function
RNA that encodes proteins
transcribed from protein-coding genes
protein synthesis
Types of RNA
Transfer RNA (tRNA)
RNA that functions as an adapter between mRNA and amino acids
selects amino acids and hold them in place on a ribosome
protein synthesis
Types of RNA
Ribosomal RNA (rRNA)
RNA that forms the ribosome, the main machinery for protein synthesis
protein synthesis
Types of RNA
Small nucleolar RNA (snoRNA)
function
RNA that facilitates chemical modification of RNAs
regulatory functions
Types of RNA
MicroRNA (miRNA)
function
- regulate gene expression by blocking translation of specific mRNAs and causing their degradation
- > 1000 in humans
- regulate >1/3 of all human protein coding genes
- base pair with specific mRNAs and fine tune their translation and stability
- guide sequences that bring destructive nuclease into contact with specific mRNA
- single microRNA can regulate a whole set of different mRNAs, as long as mRNAs carry common short sequence complementary to microRNA
regulatory functions
Types of RNA
Small interfering RNA (siRNA)
function
turn off gene expression by directing degradation of selective mRNAs and establishment of compact chromatin structures
regulatory functions
Types of RNA
Long non-coding RNAs (lncRNA)
function
- many serve as scaffolds
- regulate diverse cell properties eg. X-chromosome inactivation
regulatory functions
small nuclear RNA (snRNA)
Functions in various nuclear processes (eg. splicing)
directs splicing of pre-mRNA as part of maturation process
Prokaryotic transcription
transcription regulators
- protein that recognizes cis regulatory elements
- binding event triggers reactions that specify which genes in cell are transcribed and at what rate
Prokaryotic transcription
cis regulatory elements
- specific sequence of DNA, typically 5-10 nucleotides
- on same chromosomes as genes they control
- usually dispersed throughout genome
- Transcription of each gene controlled by its own complex arrangement
Eukaryotic transcription
RNA polymerases
genes transcribed
RP I & III: tRNA, rRNA, various small RNA
RP II: most genes, including all those that encode proteins and many non-coding genes like micro RNA, siRNA, long non coding RNAs, and most small nuclear RNA genes
Eukaryotic transcription
General transcription factors
features
- needed at almost all promoters used by RNA Polymerase II
- consist of set of proteins noted arbitrarily as TFIIA, TFIIB, TFIIC, TFIID, etc.
Eukaryotic transcription
General transcription factors
assembly - purified DNA
- TFIID binds to TATA box with TATA binding protein (TBP) subunit
- TFIID binding causes large distortion in TATA box DNA
- Other factors and RNA polymerase II assemble to region to form complete transcription initiation complex.
- TFIIH with helicase subunit unwinds DNA to expose template strand.
- Phosphate groups added by TFIIH to tail of RNA polymerase (C terminal domain / CTD)
- Polymerase disengages from general transcription factor cluster
- RP II tightens interaction with DNA and acquires new proteins that allow long distance transcription
- Once elongation begins, most of general transcription factors are released from DNA
TATA box
eukaryotes
- short double strand DNA sequence primarily composed of T and A
- typically located about 25 nucleotide upstream of transcription start site
Transcription in eukaryotic cells
process
- transcriptional activators bind to specific sequences in DNA (enhancers that can be thousands of bp away) that help attract RP II to transcription start position
- mediator coordinates proteins at promoter region
- chromatin remodeling complex and histone modifying enzyme recruited to increase access to DNA in chromosome
Eukaryotic transcription
mediator
- large protein complex that coordinates assembly of all proteins at promoter region
- allows activator to communicate properly with RP II and general transcription factors
3’ poly A tail
CPSF & CSTF
proteins involved in mRNA 3’ end cleavage
- (CPSF) Cleavage and Polyadenylation Specificity Factor
- (CSTF) Cleavage Stimulation Factor
Gene to protein overview
Eukaryotes vs Prokaryotes
Eukaryotes:
-mRNA transcribed by RNAP II
- 5’ capping, splicing, and polyA tail added in nucleus
- mature mRNA sent to cytoplasma for translation
- steps occur concurrently
Prokaryotes:
- 5’ and 3’ ends produced during transcription
- transcription and translation occur in common compartment
- translation often begins before mRNA synthesis has completed
microRNA
formation/processing
- 5’ capped and poly-A tailed
- precursor forms double-stranded structure by base pairing with itself
- cap and tail cropped off in nucleus and exported to cytosol
- cleaved by dicer enzyme to remove loop -> 23 nucleotides double-stranded miRNA
microRNA
mRNA processing
- miRNA associates with proteins to form RNA induced silencing complex (RISC)
- Argonaute protein and RISC components associate with both strands of microRNA, then cleaves and discards one strand
- argonaute holds 5’ end of miRNA and guides RISC complex to complementary mRNA 3’ UTR (>7 bases)
-
If extensive base pairing: mRNA cleaved by argonaute protein to remove PolyA tail and expose to exonuclease
- RISC and the associated microRNA released - if less base pairing: inhibition of translation, mRNA destabilization, and eventually mRNA degradation
