1
Q
Beadle and Tatum’s experiment
A
- bombarded bread mold Neurospora crassa w/ X-rays to cause genetic changes, then looked among the survivors for mutants that differed in their nutritional needs from the wild type
- wild type can grow in the lab on a simple sol’n of inorganic salts, glucose, and the vitamin biotin, incorporated into AGAR, a support medium (minimal medium)
- nutritional mutants were grown on a complete growth medium, which consisted of minimal medium + all 20 amino acids + other nutrients
2
Q
codon start/stop
A
UAA stop
UGA stop
UAG stop
AUG start / methionine
3
Q
reading frame
A
On an mRNA, the triplet grouping of ribonucleotides used by the translation machinery during polypeptide synthesis.
4
Q
promoter
A
- the DNA sequence where RNA polymerase attaches and initiates transcription
- includes within it the transcription START POINT, the nucleotide where RNA synthesis actually begins
- typically extends several dozen or more nucleotide pairs upstream from the start point
- typically includes a crucial sequence called a TATA box (TATA is the sequence on the nontemplate strand)
5
Q
transcription unit
A
A region of DNA that is transcribed into an RNA molecule
6
Q
transcription initiation complex
A
- a collection of proteins called TRANSCRIPTION FACTORS mediate the binding of RNA polymerase and the initiation of transcription; only after transcription factors are attached to the promoter does RNA pol II bind to it
- the whole complex of transcription factors + RNA pol II
7
Q
RNA polymerase and types
A
- pries the two strands of DNA apart and joins together RNA nucleotides complementary to the DNA template strand
- unlike DNA pols, RNA pols are able to start a chain from scratch, no primer needed
- binds in a precise location and orientation on the promoter, therefore determining where transcription starts and which of the two strands of the DNA helix is used as the template
- bacteria: a single type of RNA pol that synthesizes all RNA types
- eukaryotes: at least 3 types; the one used for mRNA synthesis is called RNA pol II
8
Q
transcription elongation
A
- RNA pol exposes about 10 to 20 DNA nucleotides at a time for pairing w/ RNA nucleotides
- rate: 40 nucleotides per second in eukaryotes
9
Q
transcription termination in bacteria
A
- transcription proceeds thru a terminator sequence in the DNA
- the transcribed terminator (an RNA sequence) functions as the termination signal, causing the polymerase to detach from the DNA and release the transcript
- the transcript requires no further modification before translation
10
Q
transcription termination in eukaryotes
A
- RNA pol II transcribes a sequence on the DNA called the POLYADENYLATION SIGNAL sequence, which codes for a polyadenylation signal (AAUAAA) in the pre-mRNA
- this is also the 3’ UTR
- then, at a point 10 to 35 nucleotides downstream from the AAUAAA signal, proteins associated w/ the growing RNA transcript cut it free from the polymerase, releasing the pre-mRNA
- the pre-mRNA then undergoes processing
11
Q
RNA processing
A
- in eukaryotes, the modification of pre-mRNA before the mRNA is dispatched to the cytoplasm
- both ends of the primary transcript are altered
- certain interior sections of the RNA molecule are cut out and remaining parts spliced together
12
Q
alteration of mRNA ends
A
- 5’ end is synthesized first; it receives a 5’ cap, a modified form of a guanine nucleotide added onto the 5’ end after transcription of the
- at the 3’ end, an enzyme adds 50 to 250 more adenine nucleotides, forming a POLY-A TAIL
- 3’ and 5’ UTRs (untranslated regions) are on the interior side of the altered ends; the 3’ UTR is the polyadenylation signal
13
Q
function of altered mRNA ends
A
- facilitate the export of the mature mRNA from the nucleus
- help protect mRNA from degradation by hydrolytic enzymes
- help ribosomes attach to the 5’ end once the mRNA reaches the cytoplasm
14
Q
RNA splicing numbers
A
- avg length of a transcription unit along a human DNA molecule is about 27,000 np
- however, it only takes 1,200 nucleotides in RNA to code for the avg-sized protein of 400 AA
15
Q
introns
A
intervening sequences; noncoding segments of nucleic acid that lie btwn coding regions
16
Q
exons
A
- eventually expressed, usually by being translated into AA sequences
- exceptions include the UTRs of the exons at the ends of the RNA, which make up part of the mRNA but aren’t translated into protein
- exons are sequences of RNA that exit the n ucleus
17
Q
snRNPs
A
- particles called small nuclear ribonucleoproteins that recognize the signal for RNA splicing, a short nucleotide sequence at each end of an intron
- located in the nucleus; RNA + protein molecules
- the RNA particle is called a small nuclear RNA, about 150 nucleotides long
18
Q
spliceosome
A
- several diff snRNPs + additional proteins
- almost as big as a ribosome
- interacts w/ certain sites along an intron, releasing the intron, which is rapidly degraded, and joining together the 2 exons that flanked the intron
- snRNAs catalyze these processes, as well as participating in spliceosome assembly and splice site recognition
19
Q
ribozymes
A
- RNA molecules that function as enzymes
they’re able to do so b/c - b/c RNA is single-stranded, a region of an RNA molecule may base-pair w/ a complementary region elsewhere in the same molecule, which gives the molecule a particular 3D structure
- like certain AA in an enzymatic protein, some of the bases in an RNA contain functional groups that may participate in catalysis
- the ability of RNA to H-bond w/ other nucleic acid molecules adds specificity to its catalytic activity
20
Q
alternative RNA splicing
A
- many genes are known to give rise to 2 or more diff polypeptides, depending on which segments are treated as exons during RNA processing
- b/c of this, the number of diff protein products an organism produces can be much greater than its number of genes
21
Q
domains
A
- proteins often have a modular architecture consisting of discrete structural and functional regions called domains
- 1 domain might include the active site, while another might allow the enzyme to bind to a cellular membrane
- in quite a few cases, diff exons code for the diff domains of a protein
22
Q
exon shuffling
A
- introns increase the probability of crossing over btwn the exons of alleles of a gene, simply by providing more terrain for crossovers w/o interrupting coding sequences
- while most changes would be nonbeneficial, occasionally a beneficial variant might arise
23
Q
tRNA molecule
A
- single RNA strand only about 80 nucleotides long (compared to the hundreds of nucleotides for most mRNA molecules)
- b/c the presence of complementary stretches of nucleotide bases that can hydrogen-bond to each other, this single strand can fold back upon itself and form a molecule w/ a 3D structure
- 2D: looks like cloverleaf – 4 base-paired regions, 3 of which are loops
- 3D: roughly L shaped
- loop at one end of the L: anticodon
- other end (just a strand, NOT looped): 3’ end, attachment site for amino acid
24
Q
first instance of molecular recognition
A
- matching up of tRNA and amino acid
- aminioacyl-tRNA synthetases, a family of related enzymes; 20, one for each AA
- their active sites fit only a specific combination of amino acid and tRNA
1. synthetase’s active site binds the amino acid and ATP
2. ATP loses 2 phosphate groups and bonds to the AA as AMP
3. appropriate tRNA covalently bonds to amino acid, displacing AMP
4. the resulting aminoacyl tRNA, charged w/ AA, is released by the enzyme
25
second instance of molecular recognition
- matching up of tRNA anticodon and mRNA codon
- some tRNAs are able to bind to more than one codon b/c there are 61 codons and only 45 anticodons
- wobble
26
wobble
- rules for base pairing btwn the 3rd nucleotide base of a codon and corresponding tRNA base are relaxed
- the nucleotide base U at the 5' end of an anticodon can pair w/ either A or G in the 3rd position (3' end) of a codon
- wobble explains why the synonymous codons for a given amino acid most often differ in their 3rd nucleotide base, but not in the other bases
anticodon 3'-UCU-5' can pair w/ codon 5'-AGA-3' or 5'-AGG-3'
27
ribosomes
- facilitate the specific coupling of tRNA anticodons w/ mRNA codons
- large subunit + small subunit; each subunit made of proteins and one or more rRNAs
- large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule
- catalyzes the formation of peptide bond
28
ribosome mass breakdown
1/3 proteins. the rest is rRNAs, either 3 molecules (in bacteria) or 4 (eukaryotes). rRNA is the most abundant type of cellular RNA
29
ribosome sites
- has a mRNA binding site, and 3 others
- P site (peptidyl-tRNA binding site) holds the tRNA carrying the growing polypeptide chain
- A site (aminoacyl-tRNA binding site) holds the tRNA carrying the next amino acid to be added to the chain (to the carboxyl end)
- E site (exist site) where discharged tRNAs leave the ribosome
- as the polypeptide becomes longer, it passes thru an exit tunnel in the LARGE subunit; when the polypeptide is complete, it's release thru exit tunnel
30
initiation of translation
- a small ribosomal subunit binds to both mRNA and a specific initiator tRNA (Met)
- the union of these three is followed by the attachment of a LARGE ribosomal subunit, completing the TRANSLATION INITIATION COMPLEX; all these components are brought together by proteins called INITIATION FACTORS
- the cell expends energy obtained by hydrolysis of a GTP molecule to form the initiation complex
- when it's done, the initiator tRNA sits in the P site, and A site is vacant, ready for the next aminoacyl tRNA
31
directionality of polypeptide synthesis
always in one direction. N-terminus (Met) to C-terminus.
32
translation initiation: bacteria vs eukaryotes
- bacteria: the small subunit can bind mRNA and tRNA in either order; it binds the mRNA at a specific RNA sequence, just upstream of the start codon, AUG
- eukaryotes: initiator tRNA binds to small subunit first. then, small subunit binds to the 5' cap of the mRNA and then scans downstream along the mRNA until it reaches AUG. the initiator tRNA then binds to AUG.
33
translation elongation
1. aminoacyl tRNAs anticodon pairs w/ codon at A site. (GTP hydrolysis increases the accuracy and efficiency of this step.) many different aminoacyl tRNAs are present, but only the one w/ the appropriate anticodon will bind and allow the cycle to progress.
2. rRNA molecule of the large ribosomal unit catalyzes the formation of a peptide bond btwn the amino group of the new AA in the A site and the carboxyl end of the growing polypeptide in the P site. the polypeptide chain is removed from the tRNA in the P site and is attached to the amino acid on the tRNA in the A site
3. the tRNA in the A site is moved to P site; tRNA in P site is moved to E site, where it's released.
- GTP hydrolysis required
34
translation termination
- translation continues until stop codon in mRNA reaches A site
- a RELEASE FACTOR, a protein shaped like an aminoacyl tRNA, binds directly to the stop codon in the A site. the release factor causes the addition of a water molecule instead of an AA to the polypeptide chain. this rxn hydrolyzes (breaks) the bond btwn the completed polypeptide and the tRNA in the P site, releasing the polypeptide thru the exit tunnel.
- 2 GTPs hydrolyzed to break down the translation assembly
35
polyribosomes
a number of ribosomes trail along the mRNA. enable the cell to make many copies of a pollypeptide very quickly.
36
insulin
first synthesized as a single polypeptide chain but becomes active only after an enzyme cuts out a central part of the chain, leaving a protein made up of 2 polypeptide chains connected by disulfide bridges.
37
signal peptide
- polypeptide synthesis always begins in the cytosol. there the process continues to completion, unless the growing polypeptide itself cues the ribosome to attach to the ER.
- the polypeptides of proteins destined for the endomembrane system or for secretion are marked by a signal peptide, which targets the protein to the ER
- a sequence of about 20 amino acids at or near the leading end (N-terminus) of the polypeptide; recognized as it emerges from the ribosome by a protein-RNA complex called a SIGNAL-RECOGNITION PARTICLE (SRP)
- SRP functions as an escort that brings the ribosome to a receptor protein built into the ER membrane
38
signal mechanism for targeting proteins to ER
1. polypeptide synthesis begins on free ribosome
2. SRP binds to signal peptide, halting synthesis momentarily
3. SRP (w/ ribosome + polypeptide chain) binds to a receptor protein in the ER membrane; this receptor is part of a translocation complex
4. SRP leaves. polypeptide synthesis resumes. as the chain grows, it is simultaneously translocated across ER membrane.
5. signal-cleaving enzyme cuts off signal ppeptide.
6. complete polypeptide leaves ribosome and folds into final formation
39
sickle-cell disease
mutation of a single nucleotide pair in the gene that encodes the beta-globin polypeptide of hemoglobin.this change leads to production of an abnormal protein.
wild type template strand: CTT
mutant: CAT
40
missense mutation
A nucleotide-pair substitution that results in a codon that codes for a different amino acid. may have little effect b/c the new AA could have similar properties
41
nonsense mutation
A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein.
42
spontaneous mutation
- incorrect nucleotide added to a growing chain during replication is not proofreaded
- the incorrect base will be used as a template in the next round of replication
43
gene expression: bacteria vs archaea vs eukarya
- bacterial and eukaryotic RNA polymerases are very diff. the single archaeal RNA pol resembles the 3 eukaryotic ones.
- A,E have complex set of transcription factors. B have only a small set of accessory proteins.
- A's ribosome size is similar to that of B. A, like E, share similar sensitivies to chemical inhibitors.
- initiation of translation is more similar btwn A and B.
