exam #2 Flashcards
gene
-segments of chromosomes that code for proteins
-part of a larger genome that contains all genes
prokaryotes general gene structure
-intergenic region: region between genes; varies in size and helps protect coding area
-5’ UTR: contains promoter and TSS
-start codon
-coding region
-stop codon
-terminator: can also control transcription
-3’ UTR
-intergenic region
function of intergenic region in prok
region between genes; varies in size and helps protect coding area
prokaryote promoter sequences
-35: TTGACA
-10: TATAAT (TATA/ pribnow box)
how to improve efficiency of transcription in promoter
-consensus sequences of -35 and -10
-removing a nucleotide between -35 and -10
+1 region of prokaryotes
-TSS/ transcription start site
-1st nucleotide is the first nucleotide of transcript
genome size does not =
genetic complexity
promoter function of eukaryotes
-tells transcription machinery to transcribe the region
-many promoters in euk, each has different function
-absolutely necessary to get transcription
parts of euk promoter
-TATA box
-Inr (initiator) elements
-TFIIB recognition elements (BRE)
-CAAT box
-GC elements
core promoter elements function
bind general transcription factors
euk: general promoter vs other promoters
-general: attracts machinery
-other: tells what genes to turn on for cell differentiation
introns
-noncoding sequences of a gene
-contain regulatory sequences that control gene expression and allow cell differentiation
-over 90% of human gene
discovery of introns
adenoviruses: saw that a single mRNA could hybridize from many sections of the genome and was made from blocks of sequences of dif parts of DNA
exons
-coding sequences in a gene
-can code for multiple regions
-only 3% of a gene
general transcription factors structure and function
-5 minimum required by RNA poly 2 for initiation of transcription
-elements of the promoter
TATA box location, prevalence, and what does it resemble
-only in 10-20% of RNA poly 2 targeted promoters
-upstream of TSS
-resembles -10 region of prok
gene specific transcription factors function, location, and components
-control expression of individual genes and regulate gene expression
-enhancer (distal control element)
-proximal control elements
-located upstream of promoter
enhancers of euk
-distal (far away) control elements
-binding site for activator protein
-promotes transcription
RNA nucleotide structure
has 2nd OH at 2’ C
what are consensus sequences, and how do ppl check the function of one?
-Common sequences of regions of genes that are similar across many genes
-Wreck and check: mutate specific region and see if transcription is altered
-See if mutants transcribe better or worse when promoter sequences match more closely
RNA polymerase general function, direction, error rate, facets
- adds NTPs to DNA template
-goes 5’ to 3’
-does not need a primer to start
-no proofreading activity
-no 5’ exonuclease activity
-error rate: 1 in 10^4- 10^5 nt
is the error rate of RNA poly an issue and why
Not a problem:
-Avg transcript length is short (1-3 kb or 10^3)
-Many transcript copies per gene
-RNA is not genetic material, just a temporary message made to code for the protein)
NTPs abbreviation
ribonucleotide triphosphates
holoenzyme
-in prok
-RNA poly + sigma factor
subunits of prok RNA poly and their general functions
-alpha
-omega (lower case w thing)
-beta and beta prime: bind DNA and contain catalytic active sites
-sigma: identifies correct site to start, binds to promoter
sigma factor function, consensus, most common type, action
-Required for finding where to start (promoter recognition protein)
-Promoter sequence highly conserved in bacteria
-Most common: sigma^70
-Binds between -35 and -10, anchors RNA polymerase, creates closed-promoter complex
-Polymerase unwinds 12-14 bases -> forms open promoter complex, RNA polymerase adds free NTPs complementary to antisense strand at TSS
-After ~10 NTPs added, sigma released
how many RNA polys do euk have
3 (not including mitochondrial and chloroplast ones)
RNA poly 1
-rRNAs: 28S, 18S, 5.8S
-tRNAs
RNA poly 2
-mRNAs, some snRNAs, miRNAs, lncRNAs
-transcribes protein coding genes
-the RNA poly we look at
RNA poly 3
-tRNAs, some snRNAs
-rRNAs: 5S
how many subunits do all three RNA polys of euk have
9
initiation of transcription in prok
-sigma factor binds between -35 and -10
-anchors RNA poly to create closed-promoter complex
-poly unwinds 12-14 bases to form open promoter complex
-RNA poly adds free NTPs complementary to antisense strand at TSS
-after about 10 NTPs added, sigma is released
importance of region between -35 and -10 in prok
signals location and direction of transcription
initiation of transcription in euk
- formation of TFIID: contains TATA-binding protein and TAFs/TBP associated factors
- TBP part of TFIID binds to TATA box of promoter
- recruits TFIIB to bind to TBP and consensus seq in promoter
- RNA poly recruited to promoter
- binds to TFIID-TFIIB complex in association w TFIIF
- TFIIE and TFIIH recruited, forms pre-initiation complex
- TFIIH helicase activity- unwinds DNA around TSS; kinase activity: phosphorylates C-terminal domain of RNA poly , creating conformational change in CTD and makes a strong clamp of RNA poly over DNA (need energy from ATP, etc to do this)
- conformational change causes disassociation of most GTFs from pre-initiation complex
- transcription begins
mediator in euk
-More than 20 subunits
-Interacts w gene-specific TF’s
-Stimulates TFIIH’s kinase activity and CTD phosphorylation
elongation process, direction of synthesis, orientation of RNA and DNA
-RNA polymerase associated w DNA
-Maintains unwound region of ~15 bp
-Builds a molecule thats 5’-3’, so it reads off sense (top) strand and builds off of the antisense (bottom) strand
-Region between beta and beta prime subunits contains polymerase active site -> creates covalent bonds between 3’ hydroxyl and 5’ phosphate groups of NTPs
RNA coming out of RNA polymerase: end sticking out is 5’
termination of transcription in prok
-Termination signals vary from gene to gene
-E coli: GC rich site followed by 7 A residues
-A-T bonds weaker and facilitate dissociation
-Inverted repeat of RNA transcript forms stem-loop structure (hairpin)
-Stable structure bc G-C form 3 H bonds
-Many G-C bonds on straight section
-Loop causes RNA poly to fall off
termination of transcription in euk
-Polyadenylation signals
-Phosphorylated amino acid at 3’ end causes CTD to be phosphorylated
-Recognized by RNA endonuclease and cleaved, releasing the mRNA
-5’ to 3’ exonuclease degrades remaining newly made RNA after it was cleaved from endonuclease, and the RNA poly is dislodged from the DNA template
RNA processing in prok
RNAs can be used right away due to no introns
(except for rRNAs and tRNAs)
where does RNA transcription and processing occur in euk
nucleus
what mediates RNA processing
RNA polymerase
structure/ process of adding of 5’ cap
-7-methyl-guanosine at very top
-Guanylyltransferase associated with RNA poly 2 CTD adds nucleotide in reverse orientation to 5’ end of RNA
-Guanylyltransferase ensures that each mRNA is capped as its transcribed
-Dissociates once cap is added, then cap-binding complex/ CBC binds
-Methyl group added to G residue
-5’-5’ triphosphate bridge between cap and nt of primary transcript
function of 5’ cap
-Ribosomal binding in translation
-Stabilizes and protects RNA from 5’ exonuclease
-Transport of mRNA to cytoplasm
structure/ process of adding of 3’ tail
-50-250 nt
-Usually consists of about 200 A’s
-AAUAAA: upstream of where pre-mRNA is cleaved (usually a CA sequence)
-Cleaved by endonuclease/ clipping enzyme at CA sequence
-Then poly-A polymerase adds the tail
-PABP prevents tail degradation
role of PABP
prevents 3’ tail degradation
function of 3’ tail
-Stabilizes mRNA from exonuclease
-Helps in transport of mRNA to cytoplasm
-In egg cells: helps anchor free floating mRNA
-Helps regulate (control when and how) translation of mRNA
-Changes in tail length can control mRNA translation
alternative splicing functions
-Production of different mRNAs from the same gene
-Allows for differentiation in different tissues, creates genetic diversity, can also lead to disease if created proteins are not normal/ are altered
-Multiple proteins are possible from one gene
function of splicing
removal of introns