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