Download Lecture 21-23

Adapted from Carley Karsten
Bio93 Discussion
TA: Alberto Lopez
e-mail: [email protected]
Lecture 21-23 Study Guide
I have organized some terms and topic that I think are important. This does not mean that
other topics mentioned during lecture or in the book will not be tested. This guide is meant
to clarify and emphasize certain points, NOT to list everything you need to know. I will focus
on tying things together across lectures, and giving real-life examples of the biological
principles that we are learning. Details that I include that I think will be helpful, but that you
don’t need to know, I will write in green. Questions to think about I will write in blue.
Lecture 21: Transcription
Central dogma: DNA  RNA  protein
Transcription
1. Initiation:
a. promoter = DNA sequence that indicates where the coding region of a gene
begins, and tells RNA polymerase which strand is the template strand
i. TATA box: A/T-rich region upstream of the promoter that aids in the
separation of DNA strands
What is the benefit of having lots of As and Ts here?
ii. transcription factors: in eukaryotes, these are proteins that cluster at
the promoter. Without them, RNA polymerase won’t bind. More on
this in lecture 23.
b. RNA polymerase attaches to promoter and separates the two strands of DNA
c. begins synthesizing a new strand of RNA complementary to the DNA
d. RNA, like DNA, is synthesized 5’ to 3’
Compare RNA polymerase and DNA polymerase in terms of how they
function, the requirement for a template and primer, the direction of
synthesis, and the type of nucleotides used.
2. Elongation: RNA polymerase continues to synthesize RNA… as the RNA strand
elongates, it peels away from the DNA, and the DNA double helix re-forms.
Remember, RNA is single stranded.
3. Termination: RNA polymerase reaches the end of the coding DNA, where there is a
terminator sequence. This signals to the RNA polymerase to stop transcribing, and
then fall off of the DNA.
4. all thymines are replaced by uracil (you will never see T in an RNA sequence, and you
will never see U in a DNA sequence!!)
Post-transcriptional modifications
1. 5’ end: GTP cap. This modified guanine nucleotide has an extra phosphate group
attached to it, making it slightly more reactive so that it interacts with the ribosome
better.
2. 3’ end: poly-A tail. This long string of adenine nucleotides helps protect the transcript
from being degraded (acts like a buffer). It also helps direct the transcript out of the
nucleus and into the ribosomes.
Adapted from Carley Karsten
3. splicing: removal of introns
a. intron = noncoding region of DNA or RNA
b. exon = coding region of DNA / RNA (exons are expressed)
Alternative splicing (removing different combinations of introns and exons from a
given gene) allows for efficiency and diversity. Consider: each gene contains
about 20 times the number of base pairs necessary for a functional protein
product (because of promoters, introns, etc). So if we can stick a few different
proteins within the same coding region, we save a lot of space overall. ALSO,
being able to recombine these protein modules in novel ways gives us a way to
adapt to new environments.
The genetic “code”
1. codon: triplet code. 3 nucleotides  one amino acid.
“The genetic code is redundant but not ambiguous”: what does this mean, and why
is it important?
a. start = AUG
b. stop = UAA, UAG, or UGA
2. reading frame: since the code is read in threes, we need to add or remove base pairs
in multiples of three, or else we will change how the entire sequence is read. More
on this in lecture 22.
BIO93 IS AWESOME (normal reading frame for English grammar)
BI O93I SAW ESOME (reading frame is messed up… harder to read)
Lecture 22: Translation
Transfer RNA = tRNA
1. mediates the translation of mRNA into a polypeptide; delivers amino acids to the
ribosome as needed
2. each molecule of tRNA holds one amino acid, and an anticodon. The anticodon is
complementary to the codon that codes for the amino acid. For example, if a tRNA’s
amino acid is Met (for the start codon, 5’-AUG-3’), its anticodon will be 3’-UAC-5’.
3. charged tRNA is made by the enzyme aminoacyl-tRNA synthetase, which sticks the
appropriate amino acid onto a molecule of tRNA.
Ribosomes
1. match tRNA with corresponding complementary mRNA to create polypeptide
2. made up of protein and specialized rRNA (ribosomal RNA)
3. know the structure of a ribosome: small and large subunits, E, P, and A sites
4. polyribosomes = a string of ribosomes all translating the same strand of mRNA, one
after another.
Translation
1. Initiation:
a. 5’ end of mRNA binds to small ribosomal subunit, which is also carrying the
initiator tRNA (Met)
b. mRNA slides through ribosome until the start codon (AUG) lines up with the
initiator tRNA
Adapted from Carley Karsten
c. large subunit binds; start codon + initiator tRNA sit in the P site
d. the A site is open to accept the next aminoacyl (“charged”) tRNA
2. Elongation:
a. complementary binding between mRNA codon and tRNA anticodon occurs in
the A site
b. peptide bond forms between adjacent amino acids
c. mRNA + tRNA complex slides three bases down, and whatever lands in the E
site leaves the ribosome
What happens to tRNA after it leaves the ribosome?
d. as mRNA is read 5’ to 3’, polypeptide is synthesized from N-terminus (amino
group of amino acid) to C-terminus (carboxyl group of amino acid)
3. Termination:
a. stop codon reaches the A site
b. release factor binds to the stop codon. The release factor is shaped like a
tRNA, but doesn’t have any amino acid on it.
c. release factor adds a water molecule to the end of the polypeptide,
hydrolyzing the bond between the polypeptide and the ribosome and thus
releasing it into the cytoplasm, free at last.
Is translation an exergonic or endergonic process? Why?
Post-translational modifications
1. rough ER: protein folding, glycosylation, transportation
2. Golgi: modification of glycoproteins, transportation
3. signal peptides: part of the polypeptide that directs it to the ER or the plasma
membrane. These are often made up of a sequence of hydrophobic amino acids, the
better to insert into the ER membrane or plasma membrane.
Point mutations
1. substitutions: replacement of base-paired nucleotides with a different pair of
nucleotides
a. Silent: no phenotype effect. Due to redundancy in the genetic code, this type
of mutation changes the DNA sequence without changing the final amino
acid sequence.
b. Missense: phenotype effect varies. Code for the wrong amino acid. Could be
a little bit wrong (one hydrophobic amino acid for another) or very, very
wrong (a charged amino acid in place of a nonpolar amino acid).
c. Nonsense: BAD phenotype effect. Changes an amino acid into a stop codon,
resulting in a shortened, usually nonfunctional, polypeptide.
2. insertions / deletions:
a. multiples of three: might be ok… just have an extra amino acid or two stuck
randomly in your peptide / missing a couple amino acids.
b. multiples of anything but three: distastrous. Frameshift. Everything
downstream of the mutation is read completely wrong, resulting in:
i. completely wrong sequence of amino acids (“extensive missense”)
ii. early stop codon (“immediate nonsense”)
Adapted from Carley Karsten
Likely exam question: “Translate the following sequence…” Step-by-step guide for solving a
problem like this:
1) did they give you a DNA or RNA sequence? (is there thymine or uracil?) If they gave
you DNA, make sure you transcribe to the appropriate RNA before translating.
2) what direction is it? Remember: DNA is always read 3’ to 5’, and RNA is read 5’ to 3’
3) look for start codon. No amino acids are synthesized before the start codon, AUG,
appears.
4) look for stop codon. There may not be one, but if there is, remember that no amino
acids are synthesized after the stop codon.
Lecture 23: Regulation of gene expression
Keep in mind that all the cells in your body contain the same DNA. How is it, then, that your
skin cells are so different from your muscle cells? Because the genes are regulated
differently in each cell type!!
DNA
1. chromatin modifications:
a. acetylation of histone tails makes chromatin more flexible, allowing
transcription factors better access to DNA, thereby facilitating transcription
b. methylation of DNA impedes transcription factors from reaching DNA,
thereby reducing transcription
One important example of DNA methylation that we’ve already talked about
was with Prader-Willi Syndrome and Angelman Syndrome. In both of these
diseases, one allele was silenced by genomic imprinting via methylation.
2. transcription factors:
a. in eukaryotes, particular proteins are needed in order for RNA polymerase to
bind the promoter. Without these proteins, transcription will not occur.
Therefore, changing the amounts of these proteins will have a direct impact
on transcription of a gene.
b. activators: bind to enhancer regions to facilitate transcription
i. different genes have different combinations of control elements
within their enhancers
ii. different cell types have different activators present
RNA
1. alternative splicing: different combinations of exons are selected from the primary
RNA transcript to be translated. This allows a single gene to code for many different
proteins.
2. mRNA degradation
a. mRNA doesn’t last long in the cytoplasm, despite the 5’ cap and poly-A tail
b. the cap and tail are soon removed, allowing the nucleases present in the
cytoplasm to digest the rest of the mRNA until there is nothing left
c. different genes may have different lengths of 3’ tail, altering the rate of
degradation
3. translation initiation: can be blocked, preventing attachment of mRNA to ribosome,
and thus preventing translation from occurring
Adapted from Carley Karsten
Protein
1. glycosylation: addition of sugars destined for the plasma membrane (remember this
from earlier in the quarter? where does this occur?)
2. cleavage, folding, etc.
3. addition or removal of phosphate groups to activate or deactivate a protein
4. transportation to final location (e.g. plasma membrane)
5. degradation: ubiquitin / proteasome system
a. a protein is marked for destruction by attaching a ubiquitin “tag” to it
b. ubiquitin is recognized by proteasomes
c. proteasomes degrade the tagged protein