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
RNA splicing process (general)
-Cleavage sites:
-GT: 5’ (splice donor site)
-AG: 3’ (splice acceptor site)
-Tell where to splice
-Splice at 5’ junction
-RNA folds onto itself to a branch point (usually A) in the middle-ish area of the intron
-Forms lariat intermediate
-Cleavage at 3’ splice site and ligation of exons
-Intron completely removed, then is linearized and degraded
-Exons fused together
-Reaction catalyzed by snRNAs
splice donor vs splice acceptor sequences
donor: GT, on 5’ end
acceptor: AG, on 3’ end
components of splicesome
-snRNAs: U1, U2, U4, U5, U6; complexed w snRNPs
-proteins
snRNP abbreviation
small nuclear ribonucleoprotein particles
splicesome process
-U1 base pairs with 5’ splice site
-U2 snRNP base pairs w branch point of this complex
-U1 and U2 bind to create a loop
-Other U# snRNPs join complex, bring 5’ and 3’ ends of intron together
-Intron cut/ excised, exons ligated/ joined
-Lariat released
splicing factors function
-direct spliceosomes to correct sites
-allow anchoring of splicing machinery and ensures exons are joined in the right order
where does translation occur
outside the nucleus, in the ribosomes
peptide bonds
-catalyzed by rRNA
-formed in dehydration synthesis
amino group vs carboxyl group and their other terms
-amino: N terminus (where N is)
-carboxyl: C terminus (where OH is)
structure of tRNAs
-70-80 nuc long
-Cloverleaf structure
-CCA sequence at 3’ OH terminus of amino acid arm
-Amino acids covalently bind to A
-Anticodon arm: loop opposite of 5’ and 3’ termini
-Where tRNA binds to the codon of mRNA
-Complete 3D structure: L-shape
tRNA function
allows correct amino acid to align with mRNA and thus the creation of the correct protein
specificity of tRNAs and the charging process
-aminoacyl-tRNA synthetase
-Charges the tRNA with an amino acid
-Recognizes a specific amino acid by specific binding sites
-Amino acid activated w ATP to form intermediate
-Activated amino acid is then covalently bound to tRNA at tRNA’s 3’ terminus
-Highly specific: synthetase is unique for each amino acid
-Some tRNAs can recognize more than one codon due to nonstandard base pairing/ wobble (ex: G-U)
-Caused by base pairing of G and U in the 3rd position of the codon/ anticodon
ribosomes can be made by ______
self-assembly
large subunit of euk ribosomes and what is it made of
-called 60S
-made of 28S, 5.8S, 5S rRNAs
~46 proteins
small subunit of euk ribosomes and what is it made of
-called 40S
-made of 18S rRNA
~33 proteins
binding sites for tRNA in a ribosome
-P site: holds tRNA carrying the growing polypeptide
-A site: holds tRNA carrying the next amino acid to be added
-E site: where tRNA leaves ribosome
small subunit: holds mRNA
ribozyme activity of ribosome and how to experiment for this
-rRNA causes peptidyl transferase activity and makes peptide bonds
-To experiment: get rid of both rRNA and ribosomal proteins and see when ribosomes can maintain activity
how many codons are there for mRNA and how many are start or stop
-64 possible
-3 stop
-1 start
start codon is always
-AUG
-methionine
what positive of codons is the most variable
3rd
stop codons do not have ______
a complementary anticodon
how to determine an ORF
Need both a stretch of a frame with no early stop codons and a starting methionine (only need this at the beginning of a gene)
how many reading frames do euks have
3 positive and 3 negative
initiation of translation
-Begins at AUG
-40S subunit binds to 5’ cap (cap dependent translation) and scans mRNA for start codon
-Once AUG is found -> initiator tRNA is added (forms initation complex), binds to AUG
-Then the large subunit can join 40S
-Initiator tRNA goes to P site of ribosome via eIF’s/ initiator factors
-Initiation complex released
elongation of translation
- Codon recognition: anticodon base pairs to complementary codon; GTP hydrolysis increases the accuracy and efficiency
tRNA goes to A site of ribosome - Peptide bond formation: rRNA of 60s catalyzes bond between amino acid and carboxyl end of growing polypeptide chain
- After aa is added, polypeptide removed from P site
- Translocation: ribosome moves tRNA from A to P site, empty tRNA in P site moved to E site and is ejected
- After this, mRNA moves so it can be translated at A site
- Polypeptide moves back to tRNA at P site
elongation factors (EFs)
-Escort tRNA and its amino acid to ribosome
-Factors recycled
-Powered by GTP
termination of translation
- Ribosome reaches stop codon (UAA, UAG, UGA) at A site
- Release factors recognize signals and bind to stop codon in ribosome
- Stimulates hydrolysis (addition of water to) of bond between tRNA and polypeptide chain at P site
- polypeptide chain is released
- Ribosomal subunits and other components dissociate
contig
large section of the genome
how to tell on genome browser what is intron vs exon
-exon: black boxes
-intron: lines
how to tell what strand is being read by arrow direction in browser and why
-R->L: complementary
-L->R: template
-have to read 5’-3’
isoforms
different transcripts/ copies of the gene that were spliced differently; stacked on top of each other
TSS (genome browser)
where transcription machinery sits
how to tell where exon vs intron is based on RNA Seq data
-exon: peaks
-intron: no data
higher RNA Seq peaks=
more individuals with that mRNA sequence
order of Inr, TSS, and TATA
-TATA: furthest upstream
-Inr: middle
-TSS: furthest downstream
partial codon and how to fix it
-RNA may be spliced before a complete codon
-Need however many nucleotides are missing in the next reading frame
-#1- no nuc missing
-#2- one nuc missing
-#3- two nuc missing
cDNA
DNA copy of mature mRNA transcript
SMA cause
exon 7 splicing on SMN1
The large multi-subunit complex that links the general transcription factors to the gene-specific transcription factors is called
mediator
The DNA sequence to which an RNA polymerase binds to initiate transcription of a gene is called a
promoter
A major difference between eukaryotic and prokaryotic RNA polymerases is that eukaryotic polymerases
use a set of transcription factors to bind to and initiate transcription
Transcription is _______-dependent _______ synthesis.
DNA; RNA
The first step in the formation of a transcription complex for mRNA transcription is the binding of _______ to the TATA box
TFIID
The TATA box is similar to the _______ in E. coli.
-10 promoter sequence
RNA polymerase differs from DNA polymerase in that it
does not require a primer to initiate synthesis of RNA.
The σ subunit of E. coli RNA polymerase is necessary for transcriptional elongation (T or F)
F
Termination of transcription in E. coli is signaled by
formation of a stem-loop structure in the RNA created by an inverted GC-rich sequence followed by seven A residues
Nucleosomes are an impediment to transcription in eukaryotic cells (T or F)
T
Release of RNA polymerase II to initiate transcription appears to be the direct result of the
phosphorylation of RNA polymerase by a protein kinase.
The role of the sigma (σ) factor in prokaryote transcription is to
direct RNA polymerases to bind to different promoter regions
The regions of the DNA where RNA polymerase binds can be identified by
inhibited transcription following mutagenesis in the –35 and –10 promoter regions
Termination of translation and release of the polypeptide chain occur when
a protein release factor binds to the termination codon
Which statement is true and provides evidence that a certain component of the ribosome catalyzes protein synthesis?
a.
Ribosomes are inactive after protease digestion.
b.
Ribosomes are inactive after RNase digestion.
c.
Structural analysis shows that proteins occupy the catalytic site where peptide bonds are formed.
d.
Structural analysis shows that mRNA occupies the catalytic site where peptide bonds are formed.
Ribosomes are inactive after RNase digestion
Tissue-specific RNA editing can occur (T or F)
T
During splicing, pre-mRNAs go through an intermediate stage when they are shaped like
lariat
In translation, mRNAs are read in the _______ direction, and polypeptide chains are synthesized from the _______ ends.
5ʹ to 3ʹ; amino to the carboxyl
A poly-A tail is added to an mRNA by
poly-A polymerase, which adds A’s sequentially to the end of the transcript
Processing of RNA transcripts occurs
with tRNA, rRNA, and mRNA transcripts
The RNA components of the spliceosome are five different
small nuclear RNAs
E. coli contains about _______ different tRNAs that code for _______ different amino acids.
40; 20
Which snRNA is responsible for recognition of the 5′ splice site consensus sequence in mRNA splicing?
U1
Aminoacyl tRNA synthetases are enzymes that
attach amino acids to specific transfer RNAs
Which is snRNA not part of the spliceosome?
U3
The first step in the initiation of protein synthesis is the binding of _______ to the _______.
initiation factors; small ribosomal subunit
Messenger RNAs have varying half-lives in the cytoplasm, and those differences are usually due to differences in the sequences near the 3ʹ end of the mRNA (T or F)
T
Translation of mRNAs starts at
a site downstream of a 5ʹ untranslated region.
The signal for the addition of a poly-A tail to pre-mRNA is
AAUAAA
The 7-methylguanosine cap on mRNA is required for
initiation of translation of the mRNA.
Eukaryote mRNA processing occurs in
a.
the cytoplasm.
b.
the nucleus after completion of transcription.
c.
a complex with RNA polymerase.
d.
the Golgi apparatus.
a complex with RNA polymerase.
The first amino acid that initiates the eukaryotic polypeptide is
methionine
A process called alternative splicing of mRNA transcripts can produce mRNAs with _______ from the same gene.
(all of the above)
one different exon, more or fewer exons, completely different exons
Peptide bond formation in translation occurs by a(n)
rRNA catalyzed reaction.
