Module 9 Flashcards
What is the role of RNA pol II?
- eukaryotic
- expression of most genes
- 12 subunits
What is the role of RNA pol I?
responsible for transcribing genes that encode rRNA molecules
What is the role of RNA pol III?
responsible for transcribing the genes that encode tRNAs, 1 rRNA, and other smaller RNA molecules
The transcriptional machinery finds a gene via. . .
promoter
Regulatory sequences determine. . .
WHEN and HOW MUCH the gene will be transcribed
- can be at both + or - sides
RNA pol II requires action of _____ which are required for the expression of. . .
general transcription factors (basal)
expression of ALL genes, e.g. TFIIA, B, C, . . .
Gene regulatory proteins are also called. . .
transcription factors - each trans. factor regulates the expression of a subset of genes
What is the coding strand in transcription?
the one that is NOT being transcribed
What is the function of TBP? (TATA binding protein)
the 1st protein that binds to the promoter and recognized TATA boxes in promoters that will be transcribed
What happens when TBP binds to TATA boxes?
it puts a huge dent in the DNA
- dent might make it easier for other proteins to BIND and RECOGNIZE
- dent might make it EASIER for DNA to be SEPARATED
What is the role of the transcription factor TFIIB?
binds to TBP and brings in RNA pol II
What is the role of the transcription factor TFIIH?
unwinds DNA at promoter (helicase activity)
phosphorylates Pol II at the CTD
recruits nucleotide-excision repair proteins
What kind of nucleotide is added to the 5’ end of RNA?
the 5’ cap is a 7-methylguanosine (modified nucleotide)
Outline the main features of the 5’-cap
retains all 3 of the phosphate groups
added to the 5’ end of both the end of RNA and 7-methylguanosine to form a 5’-5’ triphosphate bridge
What is the function of the 5’-cap?
protects the end of mRNA so it doesn’t get degraded by ENDONUCLEASES
allows it to be recognized by the translational machinery
Where does the 5’-cap occur?
occurs on the CTD of the RNA pol II
when the cap is produced, it is anchored to the CTD while the rest of the RNA is being transcribed
What is a spliceosome made of?
the subunits are made of both PROTEIN and DNA
What are the subunits of spliceosome called?
snRNPs
Before post-transcriptional modification, what is the RNA called?
the primary transcript
There are recognizable sequences within introns that guide splicing. What are they?
5’ end – GU
3’ end – AG
middle – usually has A
Where does splicing usually happen?
at the CTD of RNA pol II
can also happen DURING transcription
What is a polyA tail?
a large number of adenines that are added at the 3’ end
Define reading frame
a way of dividing the sequence of nucleotides in a nucleic acid molecule into a set of consecutive, non-overlapping triplets
What are the 2 vital components of tRNA?
anticodon
amino acid arm
Describe ‘wobble’ in translation (in 3rd base of a codon)
sometimes, one tRNA can be read by more than one codon
- due to altered tRNA base
e.g. inosine can base-pair to any of U, A, or C
Describe the role of the aminoacyl-tRNA synthetases in translation
- aminoacyl-tRNA synthetases make tRNAs that are attached to amino acids via ESTER linkages
- the ester linkage forms between the carboxylic group of amino acid + 3’ OH of tRNA
How is the transcriptional START codon AUG identified in prokaryotes? (translation)
the binding of the ribosome occurs are ANOTHER sequence called the Shine-Dalgarno sequence
How is the transcriptional START codon AUG identified in eukaryotes? (translation)
the ribosome binds to the 5’cap and finds and AUG with some help from a sequence called the Kozak consensus sequence
A site of ribosome
tRNA that is BOUND to 1 amino acid will bind here
P site of ribosome
tRNA that is HOLDING the growing peptide binds here
E site of ribosome
the tRNA that is LEAVING the ribosome will bind here
Explain which chemical groups of the amino acids are interacting with other amino acids and with the tRNAs during translation.
the C-terminal of amino acid at P site interacts with the N-terminal of amino acid at A side
bound to tRNA via carboxyl group
How does the peptide bond form between the two amino acids during translation?
the amino group at A site attacks the carboxyl at P site, thus breaking the bond b/w tRNA & amino acid at P site
forms a peptide bond b/w the amino group and carboxyl group
Describe the structure of a release factor
3 domains, similar to tRNA in shape
What is the role of a release factor and how does it lead to the termination of translation?
- release factor (protein) will bind to the A site at the STOP codon
- can catalyze the hydroxylation of the carboxyl end to break it off the final tRNA
- everything then dissociates to completion
Does a tRNA bind to the STOP codon?
NO
What is polycistronic mRNA?
an mRNA with multiple genes whose expression is also controlled by a single promoter & a single terminator
= operon
All eukaryotes are _____
monocistronic
Why are transcription and translation linked in prokaryotes?
Because there is no nucleus and it’s happening in the same place (doesn’t need to be transported into the cytoplasm)
Why would some ribosomes be in the rER?
- they may need to be modified (i.e. adding sugar molecule / lipid molecule)
- later could be sent to different parts of the cell or outside the cell
Where is the signal peptide found?
it is at the start of the protein sequence for those proteins that are modified, trafficked, and secreted
What percentage of genes in the human genome are coding?
only about 1.5%
What percentage of genes in the human genome are repetitive?
long repetitive sequences ~ 8%
centromeres, telomeres
What percentage of genes in the human genome are introns?
26%
Retrotransposons are sequences that. . .
descended from retroviruses
they can no longer make coat or go OUTSIDE the cell
they can only do the ‘transcription’ sequence
they ‘move’ using an RNA intermediate
Almost 45% of our genome is made up of _____
mobile elements (transposons)
What are the 2 types of retrotransposons?
SINEs
LINEs
What is a transposon?
an active piece of DNA that can ‘move’ from place to place usually b/w genes or inside introns
DNA transposons move using a DNA intermediate
Are retrotransposons actually moving?
No, they are not really ‘moving’ but rather they are making a new copy of themselves that inserts somewhere else in the genome
What are some features of LINE elements?
- 6.1 kb LONG
- can encode REVERSE transcriptase so can move autonomously (RNA –> DNA)
What are some features of SINE elements?
- short, 350 bp
- 1.5 million copies in human genome
- do NOT encode reverse transcriptase and so CANNOT move autonomous
How do SINE elements move without reverse transcriptase?
only way they can move is if there is a LINE element producing reverse transcriptase that can then act on the RNA of a SINE element
How can mitochondrial diseases arise?
can be caused by mutations in the mitochondrial genome OR the mitochondrial genes in the nuclear genome
What do mitochondrial diseases affect?
they affect energy, development, vision, and cause seizures
Does mitochondria produce ALL of its proteins?
NO, some are also produced in the nucleus and IMPORTED into the mitochondria
RNA –> precursor protein –> imported protein
Describe the human mitochondrial genome
- circular chromosomes
- dense gene-packing
- no introns
- less tRNAs (produces its own tRNAs)
- ‘variant’ genetic code; 4 of the 64 codons encode a diff thing compared to the nuclear genome
What are the really large fat bands on the gel?
rRNA
The amount of signal is relative to . . .
the amount of STARTING DNA
- tells us how highly expressed the gene is
How can we make sure ONLY mRNA is replicated (and not rRNA, tRNA, etc.)
- only mRNA has polyA-tails
- we add an oligodT (series of T bases)
- then we add an enzyme called reverse transcriptase (RNA –> cDNA)
- then, we degrade away the mRNA (with alkali) so we’re left with a single strand of cDNA
- we add a primer and use an enzyme (DNA pol I) to generate the 2nd strand
- we end up with a double stranded cDNA
- this will make a cDNA copy of every mRNA expressed in our sample
The promoter is a _____ structure
double-stranded structure (on both strands)
Operator is found in. . .
prokaryote
Does the promoter include the operator? Or, in a eukaryote, the regulatory sequences?
promoter is a very loosely defined term, and can include promoter + operator or promoter + all regulatory sequences
What is a Western blot used for?
to determine how much the gene is being expressed (how much mRNA is being translated into protein)
