Transcription and Translation CP 35-36, 49-55 Flashcards
transcription bubble
Region where DNA double helix is temporarily opened up, thus allowing transcription to occur
coding (or antisense)
The strand of DNA that has complementary sequence to mRNA
nontemplate or sense strand
The strand of DNA equivalent in sequence to the messenger RNA (same as plus strand) - complementary to the template strand, sequence is identical to the RNA
What is the general process for how mRNA is made?
1) core enzyme forms a transcription bubble (separates DNA strands)
2) core enzyme add RNA nucleotides in 5’ –> 3’ based off template
3) new RNA anneals to template strand with H bonds between base pairs
How does RNA synthesis start?
starts with a purine (usually A) in CAT by can be CGT
How is DNA unwound during RNA synthesis?
RNA polymerase unwinds the DNA (positive supercoils)
DNA gyrase and topoisomerase I return the DNA back to its normal supercoils afterwards.
Rho-Independent terminator (in bacteria)
region of DNA with two inverted repeats separated by ~6 bases followed by a stretch of A’s - As the A’s become U’s the DNA/RNA hybrid molecule becomes unstable (2 H bonds) and falls off the template strand.
Transcriptional terminator that does not need Rho protein
Rho-dependent terminators (in bacteria)
Transcriptional terminator that depends on Rho protein
Rho binds to cytosine heavy site and attaches to the RNA moving along until it catches up to the RNA polymerase at the hairpin structure. The Rho then unwinds the DNA/RNA helix and releases the RNA.
Rho protein
protein factor needed for successful termination at certain transcriptional terminators
How is bacterial mRNA translation different than eukaryotic mRNA translation?
bacterial mRNA is translated without any processing, can undergo coupled transcription/translation
eukaryotic mRNA cap
Structure at the 5′ end of eukaryotic mRNA consisting of a methylated guanosine attached in reverse orientation
a methylated (on position seven of G) GTP
what are the three parts of eukaryotic mRNA processing?
1) cap
2) poly(A)-tail
3) intron removal
poly(A) tail
A stretch of multiple adenosine residues found at the 3′ end of mRNA
what three sequences regulate the addition of the poly(A) tail?
1) recognition sequence for the polyadenylation complex (AAUAAA)
2) the cute site cleavage for the binding factor
3) the recognition sequence for polyadenylation binding protein
polyadenylation complex (AAUAAA)
Protein complex that adds the poly(A) tail to eukaryotic mRNA
What is the general process in which the poly(A) tail is added?
1) polyadenylation complex binds to AAUAAA
2) endonuclease cuts mRNA
3) poly(A) polymerase adds 100-200 adenine nucleotides
4) poly(A) binding protein binds to poly(A) tail and cap structure
makes mRNA more stable and prevents degredation
introns
Segment of a gene that does not code for protein but is transcribed and forms part of the primary transcript
streches of intervening sequence
exons
Segment of a gene that codes for protein and that is still present in the messenger RNA after processing is complete - regions that will ultimately code for a protein
primary transcript
The original RNA molecule obtained by transcription from a DNA template, before any processing or modification has occurred - is capped and tailed before introns can be removed
splicing factors
Proteins that remove intervening sequences and rejoin the ends of a molecule; usually refers to removal of introns from RNA
recognize the exon and intron borders, cut DNA, and splice exons together
how do the three modifications occur in terms of time?
capping occurs first - poly(A) tail addition and splicing occur simultaneously
when does transcription end?
when the poly(A) tail addition releases the mRNA from RNA polymerase. the mRNA then moves from the nucleus to the endoplasmic reticulum so it can be translated by the ribosomes.
Where is mRNA made in the eukaryotes?
In the nucleus
Where does translation occur in eukaryotes?
On the ribosomes in the cytoplasm
Is there coupled transcription and translation in eukaryotes?
NO!!
translation
mRNA provides the information a ribosome needs to make proteins -> translating information carried by nucleic acids to give amino acid sequences to make proteins
triplets/codons
Group of three RNA or DNA bases that encodes a single amino acid
each triplet codes for one amino acids, but multiple codons can be translated into the same amino acid
What ribosome subunits do eukaryotic ribosomes have? What do they combine to form?
40S and 60S that combine to form 80S
universal genetic code
Version of the genetic code used by almost all organisms
transfer RNA (tRNA)
RNA molecules that carry amino acids to the ribosome
recognizes the individual codons on mRNA and carries the corresponding amino acids
clover leaf shape
Loop 1 codon Amino acid —--------------- codon Loop 2 codon
What does Eukaryotic mRNA have instead of the Shine-Dalgarno sequence?
the ribosomes actually recognizes the 5’ cap structure and the Kozak sequence
anticodon
Group of three complementary bases on tRNA that recognize and bind to a codon on the mRNA
acceptor stem
Base-paired stem of tRNA to which the amino acid is attached
What is the first amino acid in each new polypeptide
methionine
aminoacyl tRNA synthetases
Enzyme that attaches an amino acid to tRNA - specific one for each amino acid
how do the codons and anticodons bind?
the first base of the anticodon binds to the third base of the codon in the mRNA.
What differs about the first amino acid in each new polypeptide (methionine) in eukaryotes?
the methionine is not modified with a formyl group
wobble (baby)
Less rigid base-pairing but only for codon/anticodon pairing during translation
explain the wobble (baby)
the nucleotide in the tRNA is not constrained by neighboring nucleotides, non standard base pairs can be created
allows for the same tRNA to read multiple codons with the same amino acid
BODER DEF: more relaxed base pairing rules - 3rd codon and 1st anticodon
codon bias
Some organisms use only a subset of redundant codons for a particular amino acid, whereas other organisms will use a different subset. This bias can hinder expression of foreign proteins in genetic engineering
each organism has a preference as to which triplet codon is used most often for a particular amino acid.
How does tRNA know when to stop adding amino acids?
Since the tRNA does not recognize a stop codon, proteins such as release factors bind to the stop codons
What is the purpose of a release factor?
Recognizes a stop codon and brings about the release of a finished polypeptide chain from the ribosome
ribosome
The cell’s machinery for making proteins
unites mRNA with the appropriate tRNAs and catalyzes the linkage of amino acids together into a chain
What is adding successive amino acids called? What does it require?
elongation and it requires elongation factors
What is elongation?
The process of adding amino acids onto a growing polypeptide chain
what are the two subunits of prokaryotic ribosomes?
30S and 50S = a 70S ribsome
Can the E-site and A-site be occupied at the same time
NO, so the used tRNA must exit before the next tRNA enters
binding sites on the 50S subunit
Acceptor
Peptide
Exit
Shine-Dalgarno sequence
Same as RBS (ribosomal binding site); sequence close to the front of mRNA that is recognized by the ribosome; found only in prokaryotic cells
ribosome binding site where prokaryotic translation begins
anti Shine-Dalgarno sequence
Sequence on 16S rRNA that is complementary to the Shine–Dalgarno sequence of mRNA
N-formyl-methionine (fMet)
a derivative of methionine - used to initiate translation in prokaryotes
What is the process of moving tRNA’s to add sucessive amino acids?
EF-T (a pair of proteins (EF-Tu and EF-Ts)) uses a phosphate group from GTP to catalyze the addition of a new tRNA into the A-site (EF-Tu), thus converting GTP to GDP.
After this reaction the GDP is exchanged for a fresh GTP for it to repeat all over again
tRNAi (tRNA sub i)
Designation for a tRNA charged with N-formyl-methionine, which is used as the first amino acid in a prokaryotic protein
translation factors
proteins needed to recruit and assemble the component of the ribosome and translational complex
Translocation
The movement of tRNA from the P-site to the E-site (the mRNA simultaneously moves one codon sideways relative to the ribosome)
What oversees the translocation step?
EF-G
initiation factors (IF1, IF2, IF3)
Proteins that are required for the initiation of a new polypeptide chain
assemble the 30S initiation complex
Because there is no nucleus in the prokaryote how does this change transcription and translation?
Transcription and translate often occur at the same time
peptidyl transferase
Enzyme activity on the ribosome that makes peptide bonds; actually 23S rRNA (bacterial) or 28S rRNA (eukaryotic)
Polysome
Group of ribosomes bound to and translating the same mRNA
In prokaryotes, multiple ribosomes bind to the same mRNA (which forms a polysome)
what is the process of polypeptide assembly
tRNAi-fMet occupies the p-site, and second tRNA enters the A-site.
fMet then releases its tRNA and moves to the E-site. the second RNA than moces to the p-site, and the cycle keeps going.
(A third tRNA enters the A-site, and forms a bond between the 2nd and 3rd amino acid –> 2nd tRNA moves into E-site of ribosome and exits. )
