Chapter 7 Flashcards
How do cells regulate the rate at which they synthesize different proteins
cells express different genes at different rates, and each RNA can direct the synthesis of many identical proteins
structure of ribose sugar in RNA
2nd carbon attached to the hydroxyl group unlike DNA which has only an H
Base difference in DNA and RNA
RNA uses uracil instead of thymine, uracil base pairs with adenine
other functions of RNA besides translating DNA to protein
structural, regulatory, catalytic roles
what determines RNA function
3D folding of RNA by base pairing with itself because it is single stranded
messenger RNA (mRNA) function
code for proteins
ribosomal RNA (rRNA) function
form the core of the ribosome’s structure and catalyze protein synthesis
microRNA (miRNA) function
regulate gene expression
transfer RNA (tRNA) function
serve as adaptors between mRNA and amino acids during protein synthesis
other functions of noncoding RNA
RNA splicing, gene regulation, telomere maintenance
how does transcription differ from DNA replication
no H-bond formation between RNA strand and DNA template
As RNA is formed, the DNA helix rewinds behind and displaces RNA
RNA copied from limited DNA region
template vs coding DNA strands
the template strand is complementary to RNA product, used to guide synthesis
coding strand has equivalent sequence to RNA product except T is replaced with U
three phases of transcription
initiation: RNA polymerase and other proteins recognize the start of a gene
elongation: RNA polymerase extends the nucleotide chain complementary to the template DNA strand
termination: RNA polymerase recognizes the end of a gene and stops transcription
RNA polymerase direction of movement
moves 3’ to 5’ on DNA template strand (synthesize RNA 5’ to 3’)
how does RNA polymerase get the energy to catalyze formation of phosphodiester bonds
hydrolysis of ribonucleotide triphosphate
how is RNA polymerase different from DNA polymerase
does not require a primer
can not proofread
uses ribonucleotides not deoxyribonucleotides
how can a new RNA transcript start before the first one is completed on a single gene
Many molecules of RNA polymerase can simultaneously bind to DNA template and transcribe the same gene because the helix reforms directly behind RNA synthesis
how does the cell decide which DNA strand is the coding and which is the template
depends on the gene and the orientation of the promoter region (RNA polymerase moves from promoter to gene, and the template strand is the strand oriented 3’ to 5’ in that direction)
prokaryotic transcription
- RNA polymerase collides randomly with DNA and binds weakly until promoter is encountered and recognized by sigma factor (without unraveling DNA)
- RNA synthesis begins at start site and sigma factor is released; transcribes template strand until encountering termination site (termination sequence is transcribed)
- RNA polymerase and transcript release and sigma factor rebinds to RNA polymerase
promoter region
region that indicates the starting point for RNA synthesis
found upstream of the gene to be transcribed
the promoter is NOT transcribed itself
-10 sequence (promoter region)
TATA box (TATAAT)
-35 sequence (promoter region)
TTGACA
what is +1 promoter region
first gene transcribed
how does the promoter region tell RNA polymerase where transcription start site is
asymmetric sequences cause promoter polarity and indicate direction to go/which strand is template/coding
terminator in prokaryotic transcription
termination signal at the end of the gene that causes halt and release of RNA polymerase and RNA product
terminator is transcribed into RNA
how many RNA polymerases do eukaryotic cells have
3 (different RNA polymerases transcribe different RNA (coding vs noncoding))
how do eukaryotic cells recognize initiation site
assembly of numerous transcription factors with RNA polymerase at initiation site
promoter region is eukaryotes much more complex, contain several regulatory elements
name of noncoding DNA in eukaryotic cells that separates genes
introns
transcription in eukaryotes requires . . .
chromatin remodeling
RNA polymerase I (eukaroyotes)
most rRNA genes
RNA polymerase II (eukaroyotes)
all protein-coding genes, miRNA genes, genes for other various noncoding RNAs
RNA polymerase III (eukaryotes)
tRNA genes, 5S rRNA gene, other small RNA genes
general transcription factors
assemble at each promoter along with RNA polymerase before transcription can begin
(TFIID, TFIIB, TFIIH, TFIIE, TFIIF)
TFIID
binds to the TATA box sequence first via TATA-binding protein (TBP)
helps attract other general TFs
causes local DNA distortion and allows TFIIB to bind
TFIIB
follows TFIID, binds to the promoter region
causes shape/structure change serves as a landmark for subsequent assembly of other proteins at the promoter - TFIIH, TFIIE, TFIIF, polymerase (forms transcription initiation complex)
TFIIH function
pries double helix apart at transcription start point, uses ATP hydrolysis for energy input
kinase domain phosphorylates the “tail” of RNA polymerase II allowing it to begin elongation (dephosphorylated at the end of transcription when it is released)
where does transcription occur in eukaryotes
mRNA synthesized and processed in nucleus before being exported to cytoplasm for translation
When are mRNA processed
as they are being synthesized
RNA polymerase carries processing enzymes on its phosphorylated tail
three processing modifications to mRNA
capping 5’ end
polyadenylation 3’ end
splicing
(1 and 2 happen on all mRNAs destined to be mRNAs)
5’ capping
addition of guanine nucleotide with a methyl group (methyl guanosine) to the 5’ end of newly formed mRNA transcript
happens fairly quickly during transcription, after ~25 nucleotides transcribed
ensures mRNA translocation, stabilizes and promotes translation
3’ polyadenylation
adds a series of repeated adenines (poly-A tail) to 3’ end of transcript
increases stability, facilitates export to cytosol, marks for translation
Eukaryotes: 3’ end first trimmed by enzyme that cuts RNA at certain sequence in the untranslated region (UTR) followed by adenylation
RNA splicing
removing introns (intervening) and joining exons (expressed) to produce mature mRNA from pre-mRNA
After 5’ capping, during transcription
purpose of introns
contain regulatory elements
alternative splicing
multiple protein variants made by joining different exon combinations from a single gene
allows the generation of different forms of mRNA from the same pre-mRNA, produces many different proteins (isoforms)
Spliceosome
RNA-protein enzyme (ribozyme) that splices mRNA introns in the nucleus
composed of several small nuclear ribonucleoprotein particles (snRNPs) which contain small nuclear RNAs and proteins
how does the cell know which part of RNA are introns
introns contain short nucleotide sequences at the beginning and end which are recognized by snRNPs- which cleave intron/exon boundary and catalyze covalent linkage of exons
intron splicing process
Adenine (key for splicing!) in intron sequence attacks opposite intron/exon boundary to form lariat structure
-OH on 3’ end of released exon attacks other intron/exon boundary and intron lariat is released (degraded later)
spliceosome complex
enormous, 3 of the proteins are U1, U2, U6
U1 in spliceosome complex
recognizes 5’ splice site and base pairs
U2 in spliceosome complex
recognizes the lariat branch point (by A) through base pairing
U6 in spliceosome complex
displaces U1 and base pairs with intron sequence (“confirming what U1 saw”)
conformation change by interaction with U2 forms spliceosome active site
Exon junction complex
RNA binding proteins deposited on junction after splicing occurs to mark the site
RNA-binding proteins
signal export to the cytoplasm, shed in the cytoplasm, new proteins mark mRNA for translation
ex: cap-binding proteins, poly-A–binding proteins, exon junction complex
mRNA degradation
mRNA eventually degraded in the cytoplasm by ribonucleases (RNAses)
3’UTR determine lifespan
Lifespan affects protein expression
intracellular condensates
proteins involved in RNA synthesis and processing aggregate in “factories”
found in eukaryotic and prokaryotic cells
monocistronic
one gene codes for one mRNA which codes for one protein
operons
in prokaryotic cells genes are organized in clusters that are transcribed together into single mRNA
under control of same promoter
mRNA is usually polycistronic and have no 5’ cap
polycistronic
mRNAs contain the information to make more than one protein
three stop codons
UAA, UAG, UGA
do not encode an amino acid
one start codon
AUG (methionine)
tRNA structure/function
two unpaired regions
-anticodon: set of three nucleotides that bind to the tRNA through base pairing
-amino acid binding site: short single stranded region at 3’ end where amino acids that match codon are covalently attached
tRNA wobble
tRNA requires accurate base pairing only at the first position and allows some mismatch at positions 2 and 3 (allows for redundancy)
Redundancy means . . .
some amino acids have different tRNA molecules
OR
some tRNAs can base pair with more than one codon
How is a tRNA charged (amino acid added)
aminoacyl-tRNA-synthetase covalently couples to corresponding tRNA (different one for every amino acid)
enzyme recognizes amino acid and anticodon
requires ATP hydrolysis
ribosomes
molecular machines that latch onto mRNA and bind to tRNAs
made of dozens of proteins and rRNAs
large subunit catalyzes peptide bond formation in growing polypeptide chain
small subunit matches the tRNA to the codon on mRNA
Ribozymes
RNA with catalytic activity
e.g. ribosomes
polyribosomes
bacterial mRNA is not processed and translation and transcription can occur simultaneously
polyribosomes are mRNA with many ribosomes attached for translation
ribosome binding sites
the ribosome has 1 mRNA binding site and
3 binding sites for tRNA:
A: amino acid
P: polypeptide
E: exit
direction of amino acid synthesis
two ribosomal subunits come together near the 5’ end of the mRNA and translate amino acid sequence N-terminus to C-terminus
start signal/ initiator tRNA
methionine (AUG)
how is initiator tRNA different from other tRNAs
it initially binds to P site instead of A site
only tRNA that can bind to p site in the absence of large subunit
importance of start signal for translation
sets the reading frame
rate of protein synthesis depends on rate of initiation
steps of translation initiation
- initiator tRNA loaded to the P site of small subunit with translation initiation factors (TIF)
- small subunit + tRNA binds to 5’ end of mRNA (marked by 5’ cap)
- Moves/scans in 5’ to 3’ direction along mRNA until encounters AUG
- Initiation factor dissociates when AUG is encountered and large subunit binds
- Protein synthesis begins
Translation elongation
- charged tRNA enters A site by base pairing with complementary codon
- amino acid it carries covalently links to amino acid held by tRNA in P site
- Large subunit moves forward (5’ to 3’) and empty tRNA moves to E site and A site is now open
- Small subunit moves forward to realign with large subunit and empty tRNA gets ejected
new amino acids are added to a growing chain at which end
C-terminal end of the growing peptide chain
translation termination
- stop codon reached and release factor binds at A site
- Release factor alters peptidyl transferase activity, so it adds water instead of amino acid to peptidyl-tRNA
- Frees up C terminal end of polypeptide chain and ribosome dissociates
Examples of protein modifications
folding, binding of cofactors, glycosylation, phosphorylation, association w/other protein subunits
proteolysis purpose
protein degradation regulates cellular protein concentration
proteasome
degrades short lived and misfolded proteins
uses energy from ATP hydrolysis
found in cytosol and nucleus
how does ubiquitin mark proteins for degradation
- proteasomes have polyubiquitin binding site which traps proteins marked with ubiquitin
- protein fed through central cylinder (active sites of proteases) and degraded until reaching stopper, ubiquitin recycled
most common step/reaction regulated in protein production
transcription
what is thought to predate DNA and proteins in evolution and why
RNA; provides structural, genetic, and catalytic roles
can store info and catalyze rxns (by ribozymes)
tetracycline effect on bacterial protein or RNA synthesis
blocks binding of aminoacyl-tRNA to A site of ribosome
streptomycin effect on bacterial protein or RNA synthesis
prevents transition from initiation complex to chain elongation, also causes miscoding
chloramphenicol effect on bacterial protein or RNA synthesis
blocks peptidyl transferase reaction on ribosomes
cycloheximide effect on bacterial protein or RNA synthesis
blocks the translocation step in translation
rifamycin effect on bacterial protein or RNA synthesis
blocks initiation of transcription by binding to and inhibiting RNA polymerase
