Week 4 Flashcards
What are the 3 steps for Transcription?
- Initiation
- Elongation
- Termination
The top strand starting on 5’ end serves as the ______ strand while the bottom strand starting on the 3’ end serves as the template for the transcriptional process.
Coding
True or false:
The coding strand is the sense strand
True
True or false:
The non-template strand is the sense and coding strand.
True
True or false:
The template strand is the sense strand.
False - the template strand is the strand running 3’ to 5’ and is the antisense strand
True or False:
mRNA is an exact copy of the antisense strand or template strand.
False
mRNA is an exact copy of the coding strand or sense strand
For the process of Transcription, the template strand is the :
Antisense strand (runs in 3’ to 5’ direction) and the mRNA produced will run in 5’ to 3’ direction)
Which strand is the mRNA being produced off of, or is read to make the initial mRNA ?
the template strand that is read in 3’ to 5’ direction
- mRNA synthesized in 5’ to 3’ direction
- the mRNA will match the sense strand/coding strand above the one it is reading
Transcription is similar to DNA synthesis, except for which 4 things ?
- Deoxy nucleotides contain ribose as the sugar
- No primer is needed to initiate process
- Uracil is added instead of Thymine
- RNA polymerase is used instead of DNA polymerase
Genes are specific regions on a chromosome that are comprised of a : _______ region and a ______ (or promoter region)
Transcribed
Regulatory
The transcribed region is _____ of the transcriptional start.
Downstream
The transcribed region contains what 4 things:
- 5’ cap site- required for maturation of mRNA
- Translation start (AUG)
- Introns and Exons
- The poly adenylation site
The promoter region is ____ of the transcriptional start and contains regulatory elements.
Upstream
What elements does the promoter region upstream of the start in transcription contain?
- TATA box (in eukaryotes - Pribnow box in Prokaryotes)
- CAAT or GC box
- Enhancers or repressors (for eukaryotic transcription)
In eukaryotes one gene =
one protein
In prokaryotes one gene =
one mRNA that may code for many genes in tandem- polycistronic (several proteins)
What are the 4 types of RNA?
- mRNA
- tRNA
- rRNA
- snRNA
What is mRNA transcribed by ?
RNA polymerase II
What needs to happen to pre-mRNA before movement in to cytosol for translation ?
5’ capping
Addition of a poly A tail
Splicing (removal of introns)
Where does maturation of mRNA take place?
in Nucleus
True or False:
Prokaryotic mRNA does not require processing
True - transcription is more coupled to translation than in Eukaryotes
What is tRNA transcribed by ?
RNA polymerase III
Does eukaryotic tRNA require maturation?
Yes
What are the maturation steps needed for tRNA?
- Removal of introns
- Addition of the 3’ amino acid attachment site (Trinucleotide CCA)
- Folds into a clover like structure with 3 loops
- Modification of bases to generate non-conventional bases
What is the purpose of modifying to non-conventional bases in the tRNA during processing inside the nucleus?
Allows for pairing to several codons
Usually in the 3rd base pairing position “wobble hypothesis”
What are the functions of tRNA?
Transports amino acids to ribosome during protein translation
Anticodon on tRNA pairs with codon on mRNA and this determines which amino acid is added to polypeptide chain in translation
What is the difference between the 3 RNA polymerases ?
All have same mechanisms of action but recognized different promoters
What polymerase makes rRNA ?
RNA polymerase 1 and 3
What does RNA polymerase 1 make?
rRNAs 28S, 18S, and 5.8S
What does RNA polymerase 2 make ?
mRNA and small nuclear RNA (snRNA)
what does RNA polymerase 3 make ?
tRNA , 5S rRNA (poly 1 makes 5.8S rRNA), and snRNAs
What direction is RNA transcribed and synthesized ?
5’ to 3’ (same direction as in DNA replication)
During initiation for Prokaryotes, RNA polymerase combines with _____ ____ (a polypeptide) to create RNA polymerase holoenzyme.
Sigma Factor
Sigma factor in prokaryotes will recognize _____ and initiate transcription (Prinbow box).
TATA box
where is the TATA box usually for prokaryotes ?
about -10
During initiation for Prokaryotes, after the sigma factor binds to TATA box, the RNA polymerase will bind ______ and ______ DNA.
Promoters
Untwist
RNA polymerase in Prokaryotes binds loosely to the ____ promoter.
-35 or TTGACA region upstream
RNA polymerase in prokaryotes binds tightly to the ____ promoter and untwists
-10 (TATA region or Prinbow box)
What are the 2 elements upstream in the Prokaryotic Initiation for RNA transcription process?
- TATA box (about -10)
2. TTGACA box (about -35)
What 5 elements are upstream from the promoter in the prokaryotic initiation process
- Cap Site (around -2)
- TATA box (about -26 to -31)
- BRE (like Transcription factor II - beta) -needed to bind RNA polymerase II (-32 to -37 region)
- Promoter Proximal elements (can increase or inhibit transcription)
- Enhancer elements (can be far away)
What 4 things must happen for tRNA maturation?
- Removal of introns
- Addition of the 3’ amino acid attachment site
- Folds into a clover like structure
- Modification of bases to generate non-conventional bases
If the tRNA contains an I (inosine) in the anticodon at the wobble position (3), this tRNA may read codons having ___ , ____, or ____ in the third position..
A, U, or C
What does rRNA require for maturation?
- Methylation
2. Incorporation into proteins (numbered subunits)
.Does Prokaryotic RNA require maturation?
NO- used immediately in process of translation
Which rRNA size is incorporated into the 40S ribosomal subunit/
18S rRNA
Which rRNA sizes are incorporated into the 60S subunit?
28S, 5.8S and 5S are put into the 60S ribosomal subunit
What combines to make the full 80S ribosome in eukaryotes?
40S subunit and the 60S subunit
Where does the reading frame always start/ what will initiate how the frame is read?
AUG - start codon
Methionine
where is the amino acid attached to the tRNA located ?
on the 3’ end of the tail
small ribosomal subunit is recruited using initiation factors (IF) which recognizes the ______ of mRNA
5’ cap
what does the cap binding complex do ?
small ribosomal subunit + 5’ cap of mRNA
- scannes mRNA for the initial AUG needed to start translation
where does the initial tRNA carrying methionine attach on the ribosome?
to the P-site ( middle site) - only one to attach here first
what happens after methionine tRNA attached to P site ?
GTP is hydrolyzed and the IF factors that found the AUG site (cap binding complex) is released
what happens after the small ribosomal subunit is released once methionine is in the P site ?
larger ribosomal subunit is recruited forming the complete ribosome
what are the three binding locations in the ribosome ?
A, P, E
all tRNA enters through the ____ site
A
all tRNA exits the ribosome through the ___ site
E
what type of energy does translation elongation require ?
GTP
what is the peptidyl transferase reaction ?
the amino acid attached to to tRNA in the P site will be transferred to the tRNA in the A site
what is the translocation reaction in the translation elongation pathway ?
the tRNA in P site is transferred to E site and the tRNA in A site will transfer to P site
- E site tRNA is then released and the A site is open again
how is translation terminated ?
stop codon is read on the mRNA
what is a missense mutation?
nucleotide change that changes the amino acid Incorporated into the protein product
what is a nonsense mutation?
nucleotide change that introduces an inappropriate stop codon
what is a silent mutation ?
nucleotide change that does not change the amino acid Incorporated into the protein product
what is an insertion mutation ?
the introduction of an extra nucleotide(s) that can change the reading frame of the translated protein ( is not a multiple of 3 )
what is a deletion mutation?
the removal of nucleotides that can change the reading frame of the translated protein
where can proteins be translated?
in cytosolic ribosomes
in ribosomes associated with rough endoplasmic reticulum
what is the transcription rate of RNA polymerase after sigma factor is released in prokaryotes?
30 to 50 base pairs a second
in prokaryotes , RNA polymerase combined with ____ _____ (a polypeptide ) to create ________.
sigma factor
RNA polymerase holoenzyme
RNA polymerase holoenzyme in prokaryotes binds promoters on DNA and untwists it , it binds _______ to the -35 promoter , and ______ to the _____ _____.
loosely
tightly
-10 promoter
after ______ of RNA synthesis occurs , sigma factor is released and recycled for other reactions.
8-9 base pairs
true or false:
part of the new RNA strand in prokaryotes is DNA-RNA hybrid , but most RNA is displaced as the helix forms
true
are eukaryotes poly or monocystronic ?
monocystronic
what does snRNA do
forms complexes with proteins used in eukaryotic RNA processing (Exon splicing and intron removal )
what is hnRNA ? describe it
heterogeneous nuclear RNA
- mRNA immediately after transcription and 5’ cap and 3’ poly-adenaline tail cap is added
which codes , introns or Exons ?
exons
introns are removed and Exons are spliced together inside the nucleus for mRNA maturation
what is another name for the GC+TATA box for prokaryotes ?
Pribnow box - attaches right before start transcription site , it attaches to the promoter region
RNA polymerase 2 transcribes a precursor-mRNA (hnRNA) that reads a promoter that can be divided into two regions …
- short sequence called Inr(initiator)
2. TATA box - located at about -30
what is the TATA box ?
- one of two core promoter elements for eukaryotes
- around -30
- TATAAAA
- TA bonds easier to denature than GC rich bonds
what is the promoter proximal element for eukaryotic DNA ?
located upstream- about -50 to -200
“Cat box “ = CAAT
“ GC box “ = GGGCGG
true or false :
different combinations of promoter region and proximal elements like cat box and GC box occur near different genes
true
what is required by RNA polymerase in eukaryotes that is similar to sigma factor in function ?
general transcription factors
- numbered to match their RNA polymerase
- binding of GTF and RNA polymerase occurs in a set order in protein coding genes
what does splicing do ?
splicing together Exon
introns are removed
what does a SNERP protein do ?
1.form a lariat structure at first cleavage site
2. structure bends over itself, carrying the Exon over to the next Exon on other side of structure
at second cleavage site
3. cuts out intron it’s attached to
4. release intron and SNERP is recycled
what is the first cleavage site for a SNERP to cut ?
—–Exon - AG (cut) GU ——
what happens after the AG-GU is cut by U1 SNERP ?
U1 SNERP bends over backwards to connect GU —— to the Adenine on the U2 SNERP ( required for forming lariat structure
what happens after the lariat structure is formed ?
U4,5,6 join on the bound U1 and U2 that were associate by U1 with it’s GU——adenine on U2
what does U5 SNERP do ?
once bound , it will cut the intron at the next AG-GU—-intron site
after ____ to _____ nucleotides have been synthesized in eukaryotes, the 5’ end of the mRNA is capped 5’ to 5’ with a ________ nucleotide __________
20 to 30
guanine
guanylytransferase
what happens after the 5’ to 5’ end is capped with guanine by guanylytransferase?
2 methyl groups (CH3) are added to cap
-essential for the ribosome to bind to the 5’ end of the mRNA
50 to 250 _____ nucleotides are added to 3’ end of mRNA . the tail stabilizes the mRNA
Adenine
in mRNA maturation, what are the three main parts of the pre-mRNA strand
- 5’ untranslated region (5’ UTR) or leader sequence
- coding sequence
- 3’ untranslated region (3’ UTR) or trailer sequence, may contain info that signals the stability of the particular mRNA
in prokaryotes vs. eukaryotes , what do prokaryotes do differently in transcription/translation ? (3 things)
- mRNA is mature and used directly after transcription
- mRNA is translated on ribosomes before it is done being transcribed - coupled
- polycistronic - information for more than one gene in tandem
what are the three things eukaryotes do that differ from prokaryotes in translation/transcription?
- mRA must be matured and processed
- transcription and translation are not coupled
- monocistronic - amino acid sequence in mRNA for one gene per mRNA , can modify it later , but is unique
what products are transcribed from genes that do not directly make protein ?
- rRNA
- tRNA -transfer RNA
- snRNA (splicesomes and other complexes )
what RNA polymerase makes tRNA ?
RNA polymerase 3
many tRNAs go through extensive post-transcriptional modification , especially in the ______.
mitochondria
tRNA forms clover leaf structures , with _______ base pairing between regions to form 4 stems and 3 loops
complementary
loop #____ contains the _____ in tRNA , which recognizes the mRNA codons during translation .
2
anti-codon
on the tRNA precursor, where are the three main introns to be removed ?
- on top of loop #2 - cut out to expose p and OH
2. cut off introns on 5’ and 3’ end of stem with no loop (tails)
what happens in loop 2 after the tRNA has been spliced ?
modification of bases and addition of anti-codon units
what’s the last step in tRNA maturation before it leaves the nucleus ?
complementary nucleotides added to tail as the anti-codon (added’ end of the stem with no loop so there is a location for amino acid binding )
- tail will match the codon from mRNA
what is tRNA unique for with nucleotides?
modified bases on the polypeptide
- ribothymidine
- dihydrouridine
- pseudouridine
- if uracil gets methylated in tRNA it changes into thymine
what does the ribosome units for prokaryotes consist of ?
30S + 50S
what does the ribosome consist of in eukaryotes?
40S and 60S - to make 80S structure ( additional proteins involved too )
true or false :
eukaryotes have many copies of rRNA genes randomly repeated
true
what are DNA regions that code for rRNA called ?
ribosomal DNA
true or false :
all genes that are transcribed are translated
false
what are the first three steps in rRNA synthesis after transcription?
- 45S rRNA precursor and 5S RNA precursor is formed and combins with proteins within a unit
- unit is methylated and trimmed
- separates into 20S and unit and 32S/5S unit
what happens after the precursor rRNA has separated into the 20S and 32S/5S units ?
- 20S becomes 18S
- 32S/5S becomes 28S/5.8S/5S
- both units leave at this point from the nucleus
in eukaryotes, the cap at the 5’ end of mRNA binds _____ and ______ ribosomal subunit containing tRNA , mRNA is scanned for AUG start codon within the ______ consensus sequence.
eIFs
40S
Kozak
for prokaryotes, in the initiation of translation, what does the rRNA bind to and scan ?
shine-dalgamo sequence upstream of AUG binds to complementary sequence in 16S rRNA
what are the first amino acid and initiation factors for eukaryotes?
- methionine
- eIFs (12 or more)
- 80S ribosome
what is the first amino acid and initiation factors for prokaryote translation?
- formyl-methionine
- IFs(3)
- 70S ribosome (30+50)
what is the sequence at the 3’ tail of tRNA that will release OH to form pairing with various AA that don’t require normal watson-crick pairing
ACC
how many possible reading frames for mRNA ?
3
what type of mutation:
CGA to CGC
makes - Arg to Arg
silent - change that makes same amino acid
what type of mutation:
CGA to CCA
makes different amino acid
missense - bad news
what type of mutation:
CGA to UGA
arg to Stop
Nonsense - changes to a stop codon - bad news
why is DNA code called degenerate code ?
many amino acids have more than 1 triplet codon that will code for that AA
in tRNA synthesis , the attached AA needs to be charged on tRNA and uses what enzyme to link it to ACC on 3’ end tail?
aminoacyl tRNA synthase (enzyme )
how does aminoacyl tRNA synthase work ?
attaches various AA to ACC on 3’ tail
charged carboxyl group OH reacts with enzyme and ATP to make ester bond linkage
where is the AA transfered to by aminoacyl tRNA synthase ?
on either 2’ or 3’ Carbon on the Adenine of ACC with a hydroxyl on the side
what happens in translation with the charged ester bond between the AA on tRNA and in making polypeptide ?
the energy from the ester bond is used to make the peptide bond in translation
what type of energy is used to make peptide bonds in translation of polypeptide chain ?
GTP that is attached to AA on tRNA is released and energy used to form peptide bond
what type of energy is used for tRNA to enter the A site ?
hydrolysis of GTP to GDP
what two things are required to move tRNA in and out of the ribosome ?
GTP
Elongation Factor
what enzyme is used to transfer the AA in P site to link to AA in A site on ribosome ?
peptidyl transferase
what is used to shift P to E and then A to P site on ribosome ?(translocation)
eEF2 and GTP
what happens when the tRNA needs to leave the E site ?
eEF2 is released
GTP is used and turns into GDP +P
what is used in the first steps of translation , after methionine has found AUG and linked ?
eEF1A and GTP are used up (turns to GDP ) so that new tRNa can enter A site
how does the eEF1A get recharged with GTP after it loses a phosphate group after dropping off tRNA in site A
a GTP comes in and donates a P group to eEF2B then remaking it into eEF2A +GTP for use again in another tRNA
what is acetylation ?
one acetyl group as below is transfered from one molecule to another
O=C-CH3
what is ADP - ribosylation ?
addition of an ADP-ribose moitie to a protein that is often done reversibility for cell functions/ DNA synthesis / apoptosis
what is carboxylation?
addition of a O=C-OH group (COOH) to a protein
what is fatty acylation ?
addition of fatty acid to protein
= carboxylic acid with hydrocarbon chain ending in a carboxyl group
what is glycosylation?
attachment of sugar moities to AA
what is prenylation ?
the addition of hydrophobic compound to a protein or molecule
What are the possible wobble combinations for the G base ?
G can pair with C as normal or with U
What are the possible wobble combinations for I base ?
C, A, or U can pair with Inosine
What is the coding start sequence on DNA for Methionine ?
ATG (it is AUG on mRNA)
what is the coding stop sequence on DNA for transcription ?
TGA
which strand is read by the RNA polymerase and what direction is it read in?
the template strand (read in 3’ to 5’) as mRNA is synthesized in 5’ to 3’ direction (parallel to the non-coding strand above it that is not read by the RNA polymerase - called the coding strand
What is the reason there are 64 possible codons ?
There are four possible bases at each of the three codon positions (4x4x4 = 64)
