Chapter 11: DNA to Proteins --> Translation

by specifying protein synthesis

how genes specify traits

proteins

polymers of amino acids

20

number of common amino acids found in proteins

peptide bonds

bonds that connect amino acids to form polypeptide chain

polypeptide chain

peptide bonds bond together amino acids to form this

function

order of amino acids in a protein determine the protein’s ?

primary structure

order of amino acids in a protein

3

code for amino acid requires ? nucleotides

experiment to determine code

1. long strands of synthetic RNAs are made that only contained one nucleotide (poly A, poly C, poly G, poly U)

2. homopolymers are added to cell free translation system that contained all 20 amino acids where one amino acid was radioactive

3. polypeptides are precipitated, filtered, and then checked for radioactivity

4. tube in which the protein was radioactively labeled contained newly synthesized protein with the amino acid that specified by the homopolymer

homopolymer

during the experiment to determine code, the tube that was labeled with the protein that carries the radioactive amino acid, specified that that amino acid coded for the ? that was used for the experiment

codon

3 nucleotides that give code for a specific amino acid

codon

some amino acids are specified by more than one ?

degenerate

when multiple codons are equivalent to one amino acid —> code is ?

1 (AUG)

number of start codons

3 (UAA, UGA, UAG)

number of stop codons

Met (AUG) and Trp (UGG)

two amino acids that only have one codon for it

tRNAs

even though there are 64 codons, there are not 64 ? with the complementary anticodon

wobble hypothesis

first bases of the codon on tRNA obeyed pairing rules, but the third base in the codon had a more relaxed base pairing, so one tRNA anticodon ca bind with more than one codon

fixes the problem of having 64 codons but not 64 anticodons on tRNAs

proposed by Watson and Crick

reading frames

for any sequence of nucleotides there are three possible sets of codons, each starting with a different nucleotides —> causes ?

each set of codons specifies different amino acids so it is essential that the ribosome begins reading codons in the correct frame

correct reading frame

always specified by the initiation codon (start codon)

AUG (methionine)

start codon sequence

modified methionine (N-formylmethionine)

what AUG specifies within bacterial DNA

formyl

group contained in modified methionine contained in bacterial DNA that is sometimes removed after the protein is synthesized (sometimes whole modified methionine is removed) (AUG in the middle of the gene specifies normal methionine)

normal methionine

in eukaryotes, AUG always specifies ?

stop codons (UAA, UAG, UGA)

codons that do not encode amino acids and has no tRNAs with anticodons that pair with these

genetic code

almost, but not completely universal (same codons specify same amino acids for almost organisms

mitochondrial genes and bacterial DNA

organisms/genes that do not follow the typical genetic code

translation

carried out on ribosomes, with tRNAs serving to bring the appropriate amino acid for each codon

initiation, elongation and termination

3 stages of translation

binding of ribosome to mRNA and finding start codon

what causes mRNA to be translated

tRNAs with anticodon binds with each codon, brings in amino acid, and is added to growing chain

how each amino acid is added to the growing chain

methionine

AUG

in both prokaryotic and eukaryotic cells, translation always initiates with ? encoded by ?

signals

the ? that identify the specific AUG to start translation are different in prokaryotic and eukaryotic cells

Shine-Dalgarno sequence

signal that identifies initiation codon in PROKARYOTIC mRNAs

Shine-Dalgarno

part of the rRNA in the small subunit of ribosome has a sequence that is complementary to the ? sequence that allows ribosome to bind to prokaryotic mRNA

7-methylguanosine cap at the 5’ terminus

signal that identifies initiation codon in EUKARYOTIC mRNAs —> ribosomes then scan downstream of mRNA until they encounter first AUG —> translation begins

tRNAs

these are transcribed and then must be attached to the correct amino acid that corresponds to its anticodon

aminoacyl-tRNA syntheases

enzymes that recognize a particular amino acid as well as all the tRNAs that accept that amino acid

20 different enzymes (one for each amino acid) that transcribe tRNAs

tRNA charging

when aminoacyl-tRNA syntheases use ATP for energy and attach the amino acid to the 3’ end of the tRNA

anticodon

part of tRNA that base pairs with the complementary codon on the mRNA

antiparallel

binding of tRNA and mRNA are still ?

ex. codon 5’ UGG 3’ base pairs with the anticodon 5’ CCA 3’ (flipped it matches: ACC)

ribosomes

responsible for binding the mRNAs and tRNAs during translation

large and small subunits

prokaryotic and eukaryotic ribosomes both have ? and ?

RNAs and proteins

what each subunit in ribosomes are made up of

small and large subunits

these ribosomal components remain separate until translation is initiated

initiation factor-3 (IF-3)

factor that binds to small ribosomal subunit during translation and prevents large subunit from binding

Shine-Dalgarno

once IF-3 is bound to the small subunit of the ribosome during bacterial initiation of translation, the small subunit binds the mRNA and finds the ? sequence

bacterial initiation

1. initiation factor-3 (IF-3) binds to small ribosomal subunit

2. small ribosomal subunit + IF-3 binds to mRNA and finds Shine-Dalgarno sequence

3. initiator tRNA, fMet-tRNA, attaches to initiator codon, along with IF-2, IF-1, and GTP (30S complex)

4. initiation factors disassociate and GTP hydrolyzes to GDP —> allows large subunit to bind forming the 70S initiation complex

fMet-tRNA

tRNA that attaches to the initiator codon in mRNA

IF-1, IF-2, and GTP

initiator factors/energy molecule that bind to mRNA or initiator tRNA after fMet-tRNA binds

IF-1, IF-2, and GTP

initiation factors and energy molecule that forms 30s complex during bacterial initiation

70S complex

once initiation factors disassociate and GTP goes to GDP in bacterial initiation, large subunit binds to mRNA forming ? complex

5’ cap

where initiator complex binds in eukaryotic initiation

start codon

once initiator complex binds to 5’ cap in eukaryotic initiation, the complex scans the mRNA to find the ? in the middle of the consensus sequence (Kozak sequence)

Kozak sequence

consensus sequence that is in eukaryotic mRNA and contains a start codon

binding of 3’ poly(A) tail binding proteins and 5’ Cap-binding proteins

binding of the small ribosomal subunits to the 5’ cap in eukaryotic initiation is enhanced by the ?

looped

during eukaryotic initiation, the mRNA is ? so the Cap-binding proteins and poly(a) binding proteins can interact with each other

elongation

when new amino acids are brought by tRNAs and added to the growing polypeptide chain

aminoacyl (A) site

peptidyl (P) site

exit (E) site

3 ribosomal sites that can be occupied by tRNAs

P site

during initiation, the initiator tRNA immediately occupies the ? site on ribosome

A site

where all tRNAs (except initiation tRNA) binds on ribosome

elongation

1. after initiation tRNA (fMet-tRNA) binds to start codon, it occupies the P site on the ribosome

2. a charged tRNA with correct anticodon binds with elongation factor Tu (EF-Tu) and GTP, it binds to the A site

3. once charged tRNA binds to A site, GTP gets cleaved to GDP and the EF-Tu-GDP complex is released from tRNA

4. rRNA in the large subunit of the ribosome forms a peptide bond between two amino acids —> growing peptide is now on the tRNA in the A site

5. ribosome then translocates down the mRNA to position its A site over the next A site

6. as ribosome shifts down, tRNA in P site is now in E site and the tRNA in the A site is now in the P site

7. tRNA in the E site now leaves to be recharged

8. with A site open, new tRNA with correct anticodon binds and repeated until whole polypeptide is created

peptide bond

when one charged tRNA is in the P site and another is in the A site, the large subunit of the ribosome forms a ? bond between the two amino acids to start growing polypeptide chain and place it in A site

elongation factor-G (EF-G)

elongation factor that is respond for hydrolyzing GTP to GDP and translocating/shifting ribosome down mRNA to place A site over the next codon

eEF2

EF-Tu

EF-G

three elongation factors in eukaryotic elongation

EF-Tu

elongation factor that binds with GTP to A site on A site of ribosome

EF-Tu

EF-G

two elongation factors in bacterial elongation

eEF2

elongation factor that shifts ribosome down mRNA to place next codon in A site of ribosome only in eukaryotic elongation

eEF2

elongation factor that is a target of the bacterial diphtheria toxin

when toxin is present, it inhibits eEF2 and prevents translocation of ribosome along mRNA and halts protein synthesis

bacterial diphteria toxin

toxin that inhibits eEF2 which prevents translocation of ribosome along mRNA and halting protein synthesis in eukaryotic mRNA

termination

caused when ribosome translocates and a stop codon is in the A site of ribosome

protein synthesis

ends when ribosome translocates and a stop codon is in A site during translation

stop codon

once this codon is in the A site of ribosome during translation, there are no tRNAs with anticodons for it to bind to mRNA so no tRNA enters the A site

RF-1

RF-2

RF-3

3 release factors that terminate protein synthesis

RF-1

RF-2

these release factors bind to the termination codons

RF-3

this release factor binds to GTP and ribosome during termination

termination

1. polypeptide is released from tRNA in the P site

2. RF-1/RF-2. bind to the termination codons in A site

3. RF-3 binds with GTP and binds to the ribosome

4. ribosome undergoes conformational change and releases RF-1/RF-2 from A site

5. tRNA in the P site moves to the E site and leaves while GTP is hydrolyzed to GDP

6. mRNA is released and the ribosome subunits dissociate

ribozyme

an RNA with enzymatic activity

enzymatic

when rRNA in the large subunit of the ribosome forms a peptide bond during elongation, the RNA is exhibiting ? activity

untranslated nucleotides

stop codon in mRNA is followed by ?

3’ UTR (untranslated region)

untranslated nucleotides that follow the stop codon in mRNA

3’ UTR

often contain sequences that affect the stability of the mRNA and influence whether the mRNA is translated

proofreading

in bacteria, evidence supports the proposal that bacterial ribosomes engage in ? (similar to DNA polymerases)

premature termination

after translocation, the ribosome checks the pairing of the codon and anticodon at the P site, if pairing is incorrect, the mRNA and tRNA are not aligned properly —> triggers ? of translation (RF-3 assists in this function)

polyribosomes (polysomes)

when mRNA molecules are translated simultaneously by multiple ribosomes

in both eukaryotic and prokaryotic cells

as new ribosome moves down the mRNA, a new ribosome can begin translating at the beginning of the mRNA

chaperone proteins

some proteins are not able to fold into their correct tertiary structures right after translation —> these proteins are needed

modifications

after translation, proteins in both prokaryotic and eukaryotic cells can undergo ?

cleaved and trimmed

some proteins must be ? and ? to be functional, others have carbohydrates or other groups attached

acetylated

in eukaryotic cells, the amino end of a protein is often ?

antibiotics

these work by binding to bacterial ribosomes and inhibiting specific steps in translation

ONLY bacterial ribosomes not eukaryotic ribosomes

tetracyclines

bind to the A site of ribosomes and block entry of charged tRNAs

type of antibiotic

chloramphenicol

binds to the large subunit and inhibits peptide bond formation

type of antibiotic

streptomycin

binds to the small subunit of the ribosome and inhibits initiation

type of antibiotic

erythromycin

blocks translocation

type of antibiotic