Unit 4 Learning Goals
Give the abbreviations for 3 sites (A, P, E) for tRNAbinding to the ribosome; know what
each abbreviation stands for.
P site: peptidy
E site: exit site
Be able to state structural features that are shared among all tRNAs, including length (73-93
nucleotides); presence of modified bases; 3 segments of Watson-Crick base-pairing that account for
half of the structure; 3 stem-loop structures, including the anticodon loop and the TΨC loop; the
acceptor stem at the 3’end; the pG at the 5’end.
1. Each is a single chain containing between 73&93 ribonucleotides
3. 3’CCAterminal region which is part of a region called the acceptor stem and 5’
end of tRNAis phosphorylated usually pG.
4. TwC loopp which is named from the sequence ribothymine pseudoracil cytosine
5. The extra arm which contains a variable number of residues
6. DHU loop containing dihydrouracil residues
7. Anticodon loop
Know the function and location of several important sequences of tRNAs: CAA(acceptor)
stem; anticodon loop; TΨC loop.
CCAaccept stem is located at the end of the 3’that usually attaches to the activated
amino acid. This single strand region changes conformation in the course of activated amino
acid and protein synthesis.
The anticodon loop is located at the other end of the L which makes it accessible to the
TwC loop has a sequence of ribothymine pseudoruacil cytosine.
Identify locations of mRNA, small-subunit RNA, large-subunit RNA, and tRNAs inA, P, and
E sites in cartoon diagrams of ribosomes.
Given a list of steps in intiation and elongation, state which utilize GTP-hydrolyzing enzymes; and
be able to give the names of the 3 GTP-hydrolyzing enzymes.
IF1& IF3 bind to the 30S to prevent premature joining of 50S to form a dead 70S complex. IF1
binds nearAsite and directs fMet-tRNAto the P site. IF2 binds GTP and the conformation chances and binds with mRNAand the 30S subunit forms the 30S initiation complex. IF2 stimulates association of
50S subunit complex.
Elongation factor delivers aminoacyl-tRNAto the ribosome. (EF-Tu) binds aminoacyl tRNAonly
in its GTP form and releases it to the ribosome in tis GDP form.
Give the difference between the 2-D (secondary) and 3-D structures of tRNAs and recognize
cartoon drawings of each.
Know which ends of tRNAbind to small and large rRNAsubunits.
The anticodon end of the tRNAinteracts with the 30S subunit, while the other end without the
anticodon interacts with the 50S.
Identify specific roles of the small ribosomal subunit (initiation and codon readout); and the
large subunit (peptide bond formation).
3’end of rRNAcomponent of the 30S subunit contains a sequence of base
complementary to the purine rich regions of mRNA. Two kinds of interactions determine where
protein synthesis starts: the pairing of mRNAbases with the 3’end of 16s rRNAand the pairing
of the initiator codon on mRNAwith the anticodon of an initiator tRNA.
Know the 3 components of the ternary complex formed during initiation: fMet-tRNAi ; mRNA;
and the small ribosomal subunit; and how this set of 3 molecules interacts with each other
Bacteria protein synthesis is initiated by formylmethionyl transfer RNA. Transformylase
formulates the methionine+tRNA. There are three components that are formed during initiation.
The fMet-tRNAmolecule occupies the P site of the ribosome position so the anticodon pairs with
the initiation codon on mRNA. TheAand E sites are empty.
Know which proteins specifically recognize the initiator tRNAin prokaryotes: transformylase and
Know that the aminoacyl-tRNAsynthetases are the enzymes that enforce the genetic code, i.e.
match each amino acid to its set of specific anticodons.
The attachment of tRNAestablishes the genetic code.
Amino acid +ATP->Aminoacyl-AMP + PPi
Aminoacyl+ tRNA-> aminoacyl-tRNA+AMP
Two molecules of ATP is consumed in the synthesis of each aminoacyl-tRNA. One of
them is consumed in forming the ester linkage of aminoacyl-tRNA, whereas the other is
consumed in driving the reaction forward. Know which portions of tRNAmolecules are predominantly involved in recognition by
aminoacyl-tRNAsynthetases (anticodon loop and acceptor stem), but that this is not an absolute
rule and that other portions of tRNAs are sometimes more important in determining specificity.
Know which enzymes (aminoacyl-tRNAsynthetases) activates the amino acid so that the actual
peptidyl transferase step is exergonic.
Aminoacyl-tRNA synthetase are activating enzymes that activate the amino acids. The
sum of the reactions is highly exergonic Know the activated forms of the amino acid that are produced by this enzyme, including
aminoacyl-AMP(formed from the amino acid andATP; stays bound to the enzyme); and
ultimately, the ester between the amino acid and the 2’or 3’–OH group of the 3’terminal ribose.
Amino acid+ATP->Aminoacyl-AMP+ PPi
An amino acid ester of tRNAis called aminoacyl tRNA or sometimes a charged tRNA.
Know the role of EF-Tu in delivering aminoacyl-tRNAmolecules to theAsite of the ribosome,
especially the role of GTPhydrolysis in releasing the aminoacyl-tRNAto the ribosome, as well as
the role of EF-Ts in catalyzing the restoration of a new molecule of GTPto the released EF-Tu.
Elongation factor delivers aminoacyl-tRNAto the ribosome. (EF-Tu) binds aminoacyl tRNAonly
in its GTP form and releases it to the ribosome in tis GDP form. The binding of Ef-Tu to aminoacyl tRNA
protects the ester linkage from hydrolysis in addition if the anticodon is not properly paired with the
codon, hydrolysis will not take place and the aminoacyl tRNAis not transferred to the ribosome.
Know that the peptidyl transferase site of the ribosome is an RNAenzyme (ribozyme).
Peptidyl transferase catalyzes peptide bond synthesis. The formation of the peptide bond
in the 50S subunit is located deep in the 50S tunnel that allows the nascent peptide to leave the
ribosome. The peptide bond is formed from proximity and orientation. The ribosome orients and
positions the two substrates so they take advantage of the reactivities.
Know which accessory proteins couple GTPhydrolysis to which steps in translation: (IF2; EF-
Tu; EF-G; RF2)
Translocation is enhanced by elongation factor G (EF-G also called translocase). The
binding of EF-G to the ribosome stimulates the GTPase activity of EF-G. on hydrolysis, EF-G
undergoes conformational change that displaces the peptidyl-tRNAin theAsite to the P site,
which carries the mRNA and the deacylated tRNAwith it.
EF-Tu protects the aminoacyl-tRNAfrom hydrolysis and if the anticodon is not properly
paired with codon, hydrolysis does not take place and the aminoacyl tRNa is not transferred to
Know the direction of growth of the polypeptide chain (5’-3’on the mRNA, or N- to C-terminus)
The polypeptide chain grows from 5’->3’or N terminal to C terminal.
Know the approximate rate of growth of the polypeptide chain in prokaryotes (~5 amino
acids per second).
Given the structures of the two aminoacyl tRNAs bound to the peptidyl transferase site, be able to
draw electron-pushing arrows corresponding to the nucleophilic attack that forms a new peptide
P.904Amide group (Asite) attacks the carbonyl group (P site)
Know the role of RF1 in binding to theAsite and positioning a water molecule to hydrolyze the
ester linkage of the completed protein to the last tRNA.
Release factors. One of the release factors recognize UAG and RF2 are proteins that
resemble a tRNAmolecule. The RF interacts with the peptidyl transferase center.Awater
molecule attacks the ester linkage between the tRNAand the polypeptide chain freeing the
polypeptide chain. The detached polypeptide leaves the ribosome and the 70S complex
dissociates through hydrolysis of GTP.
Explain how there are two general types of polymerizations in biology, depending on where the activating group gets cleaved (from the newly-added monomer, or from the end of the
Type 1 polymerization the activating group X is cleaved from existing polymer and the
activated monomer keeps its X group. Protein synthesis is type one.
Type 2 polymerization is the activating group X is cleaved from the monomer which is
being added. DNAand RNAsynthesis are type two.
Know that in eukaryotes, initiation starts with binding to the 5’cap, and then the small subunit
of the ribosome, with initiator Met-tRNA atfached in its Psite, slides along the mRNAuntil it
finds the firstAUG codon.
In eukaryotes the initiating amino acid is methionine rather than N-formylmethionine.
Understand why eukaryotes don’t have, and don’t need, a Shine-Dalgarno sequence in initiation.
The initation codon is always AUG. instead theAUG from the 5’mRNA is usually
selected as the start site and do not have specific purine rich sequence on the 5’side to
distinguish initator AUG from internal ones.
Know the names of the antibiotics listed in this chapter that interfere with protein synthesis.
Streptomycin, Puromycin, tetracycline
Know what SRPbinds to on the growing polypeptide chain, and in the endoplasmic reticulum
The SRP binds to a sequence on the ribosome as soon as the signal exits the ribosome.
SRP shepherds ribosome and its nascent polypeptide chain to the ER membrane. SRP binds all
ribosomes that display the signal sequence.
Know the sequence of steps in synthesis of secreted proteins: Binding to SRP; transfer of
ribosome+nascent peptide to the ER; translocation of the N-terminus across the ER membrane;
signal peptide cleavage; completion of protein synthesis.
1. SRP binds to ribosome halting protein synthesis.
2. SRP-ribosome complex docks with SRP receptor in the ER membrane
3. SRP and SRP receptor hydrolyze bound GTP and protein synthesis resumes and the
SPR is free to bind to another signal.
4. Protein synthesis occurs in the ER.
Chapters 31 & 32
Know differences in relative genome sizes of prokaryotes and eukaryotes
E.coli (prokaryotes): 1 circular chromosome, 2000 genes
Yeast (simple eukaryote): 16 chromosomes, 6000 genes
Human (complex eukaryote): 46 chromosomes, 25000 gene Be able to explain how chromatin structure in the eukaryotic nucleus reduces the effective size
of the eukaryotic genome that the transcriptional apparatus must scan in order to find promoter
Winding of DNAaround the nucleosome core contributes to the packing of DNAby
decreasing its linear extent. Wrapping this DNAaround the histone octamer reduces the length around
the long dimension of the nucleosome.
Know differences in DNAorganization (nuclear organization; role of nucleosome core
particles and histones).
Nucleosomes: repeating units of the histone octamer and the associated DNA
Nucleosome core particles: smaller complex formed by the histone octamer and the DNAfragment
Epigenome: differences in chromatin structure and covalent modifications of the DNAlead to
different cell types.
Recognize 3 motifs for secondary/tertiary structure found in DNAbinding proteins: helix-turn-
helix; zinc finger; leucine zipper.
1. Helix-turn-helix(Homeodomain): form heterodimeric structures that recogonize asymmetric
DNAsequences. Each homeodomain has a helix-turn-helix motif with one helix inserted into the major
groove of DNA
2. Basic-leucine Zipper: each alpha helix is a basic region that lies in major groove of DNA&
makes contacts responsible for DNA-site recognition. Because these units are often stabilized by
appropriately spaced leucine residues, these structures are often referred to as leucine zippers.
3. Zinc-finger proteins: short protein sequences each binding an Zn ion through a
conserved motif residues in a short helix. Zinc finger domains are strung together by short
linkers and follow the major groove of DNA.Alpha-helix from each domain makes specific
contact with edges of base pairs within the groove.
Know that for expression of genes in eukaryotes, chromatin structure must be relaxed (in
specific cell types and at specific stages of development).
Euchromatin: expressed chromosomes
Heterochromatin: repressed chromosomes
Facultative: silenced only in certain context
Constitutive: obligatorily silenced
Know that DNAsequence elements that bind dimeric proteins (nuclear hormone receptors in
eukarotes; repressor proteins in prokaryotes) often involve partially palindromic sequences, with
key sequences separated by ~3.5 nm, or ~10 base pairs, matched to a similar spacing of binding
sites in the dimeric proteins.
Regulatory sizes are usually binding sites for specific DNA-binding proteins, which can
stimulate or repress gene expression. Dimeric proteins that bind to DNAsequences with inverted repeats or palindromic sequences
Know several themes for eukaryotic transcriptional regulation: (1) Enhancers; (2) nuclear
hormone receptors; (3) DNAmethylation (of cytosines).
Enchancers: region of DNAincreases the transcription factors to enhance transcription levels of
Nuclear hormone receptors: Steroids are hydrophobic molecules that can easily diffuse across
cell membranes. These receptors have similar modes of action
1. On binding of the signal molecule, the ligand receptor complex modifies the
expression of specific genes by binding to control elements in the DNA.
2. Two highly conserved domains, a DNA-binding domain and a ligand binding domain
3. These ligands need soluble carrier proteins in blood plasma, but can diffuse easily
across cell membrane by themselves
4. Once in the cell, estrogen bind t o highly specific soluble receptor proteins in the
cytoplasm that can diffuse across the membrane
5. Recruitment of coactivator catalyze reactions leading to the modification of the
DNAmethylation: inhibition of gene expression to specific cell types. Cytosine sites are
methylated. The methyl group of 5-methylacytosine protrudes into the major groove where it
could easily interfere with the binding of proteins that stimulate transcription.
Know that enhancers and nuclear hormone receptors appear to operate by regulating
Enhancers function by serving as binding sites for specific regulatory proteins. DNA
binding proteins influence transcription initiation by perturbing the local chromatin structure to
expose a gene rather than direct interactions with RNApolymerase.
Know that DNAmethylation operates by regulating access of proteins to the DNAmajor groove.
-inhibits gene expression by methyltransferases by inhibiting the acess of proteins to bind to the
Know 4 covalent modifications of histone tails: acetylation; phosphorylation;
methylation; ubiquitylation. Know which types of residue are modified (by the first
Acetylation (Lysine): attachment of an