UNIT 4 Flashcards
what are the 4 components for translation
tRNA, mRNA, ribosome, and aminoacyl tRNA synthetase
function of mRNA
consists of codons
function of tRNA
provides physical interface btw amino acids being added to growing polypeptide chain and codons
function of aminoacyl tRNA synthetase
couple amino acids to specific tRNAs that recognize the appropriate codon
function of ribosome
composed of RNA and protein, coordinated correct recognition of mRNA by each tRNA and catalyzes peptide bond formation btw growing chain and amino acid attached to selected tRNA
what is open reading frame (ORF)
protein coding region of mRNA that is composed of contiguous, non-overlapping strings of codons
what are the start codons and their order
pro: AUG>GUG>UUG
euk:AUG
codes for Met
what are the stop codons
UAA, UAG, UGA
describe how many ORF’s in pro and euk
pro-monocistronic (single)
euk-polycistronic (multiple)
components of prokaryotic mRNA
- ORF
- ribosome binding site (rbs)
- spacer
what is the ribosome binding site (rbs)
short sequence upstream of start codon that facilitate binding of the ribosome to mRNA
what is Shine-Dalgarno sequence
the ribosome binding site sequence
located on 5’ side of start codon
5’UAAGGAGGU3’ 4-8nts
purine rich
how does ribosome binding site work
complementary to Shine-Delgarno sequence is 16S rRNA
ribosome base pairs with this, aligning ribosome to beginning of ORF
what is 5’ cap
a chemical modification in eukaryotes that recruits ribosome and stabilizes mRNA (joined to 5’ end of mRNA via 5’ to 5’ linkage)
what is the Kozak sequence
purine three bases upstream of start codon, and guanine immediately downstream (most lack but those that have, increase the efficiency of translation)
what is a 3’ poly-A tail
added be enzyme poly-a-polymerase to 3’ end of mRNA, results in stabilization of mRNA and more efficient translation
how long are tRNAs
75-95 nts
what does the 3’ end of tRNA have and its function
universally conserved CCA sequence
-absolutely required for protein synthesis because that is the site that is attached to cognate amino acid
describe the unusual nucleotides found in tRNA
- post transcription
- found in all 4 bases
- more complicated modifications in purines
- some are species specific, some are tRNA specific
- modified by specific enzymes
what are the three roles of modification in tRNA
stability, sequence diversity, anticodon-codon recognition
Stability has a role in the modification of tRNA, describe it
without some of these modifications t1/2 of tRNA reduced from hours to minutes
sequence diversity has a role in the modification of tRNA, describe it
offers extra recognitions for aminoacyl-tRNA synthetases to recognize the appropriate tRNA for amino acid charging
anticodon-codon recognition has a role in the modification of tRNA, describe it
expansion of the capability of a tRNA to recognize multiple codons (eg 1 tRNA can possibly recognize 3 codons)
describe the secondary structure of tRNA
clover leaf like structure
acceptor arm: site of attachment of the amino acid, formed by pairing between 5’ and 3’ end of the tRNA molecule; contains the 5’CCA3’ sequence
D loop: made up of dihydrouridines
Anticodon loop: contains the anticodon (3 nucleotide long sequence that is responsible for recognizing the codon by base pairing with the mRNA) on it’s 3’ end it has purine and 5’ end it has uracil
variable loop: varied from 3-21 bases
TPsi loop: contains unusual base PsiU (sequence reads 5’TPsiUCG3’
Describe the tertiary structure of tRNAs
inverted L shape
Describe the 4 steps to attach amino acids to tRNA
- Recognize acceptor arm and anticodon arm
eg. Met/Trp - Recognize specific nucleotide pair in acceptor arm
eg. Ala and Cys
G3-U70 pair in acceptor arm of Ala-tRNA
C3-U70 pair on acceptor arm of Cys-tRNA
change C3-G70 to G3-U70 in Cys-tRNA switched this mutated tRNA to be charged with Ala - recognize variable loop
eg. Serine - recognize modified base
eg. Y in Phe-tRNA
describe a charged and uncharged tRNA
charged contains an amino acid
uncharged has no amino acid
What are the two classes of enzymes (tRNA synthetases)
Class I enzymes: attach amino acid to 2’OH of the tRNA (generally monomeric)
Class II enzymes: attach amino acid to 3’OH of tRNA (typically dimeric or tetrameric)
What are the function of aaRS
recognize different combinations of identity elements in tRNA
Describe the modular structure of aaRS
- catalytic domain
- tRNA recognition domain
- editing domain (may be separate subunit)
how many aaRS are there
20
Describe the 1st step rxn that is catalyzed by aaRS
Adenylation (transfer of AMP)-done via hydrolysis of pyrophosphate by pyrophosphatase
amino acid reacts with ATP to become adenylated (aminoacyl-AMP) and releases PPi (pyrophosphate)
RESULT: amino acid is attach to adenylic acid via high energy ester bond which the carbonyl group of amino acids is joined to the phosphorylated group of AMP
[amino acid+ATP->aminoacyl-AMP + PPi]
Describe the 2nd step in the rnx that is catalyzed by aaRS
tRNA charging-
adenylated amino acid (which remains tightly bond to synthease) reacts with tRNA
RESULT: transfer of amino acid to 3’ end of tRNA via the 2’ or 3’ hydroxyl and release of AMP
[amino acid+tRNA=ATP->aminoacyl-tRNA+AMP+PPi]
[PPi->2Pi]
What is the error frequency for the amino acid charging step
and what is the overall error rate
~10^-5
overall=1.67*10^-5
What is the double sieve model
proposing that an amino acid larger than the correct one is rarely activated because (1) it is too large to fit into the active site of the tRNA synthetase (first sieving), and (2) the hydrolytic site of the same synthetase is too small for the correct amino acid (second sieving). Thus, an amino acid smaller than the correct one can be removed by hydrolysis.
- Active site (AS, SS) and editing site (ES) have different shape and size
- AS fits into cognate substrate the best (Ile doesn’t fit into editing)
- Leu is too big to fit into AS but both Ile and Val can bind
- ES is too small for Ile but fits Val
Describe the proofreading function of Ile-tRNAIle synthase
- with val attached to tRNAIle, the CCA tail shuttles betwen AS and ES
- Val will be hydrolyzed from tRNAIle
- -this is the 1st reason why the 3’ end tail of tRNA has to be single stranded
describe the 2 subunits that make up the ribosome
small subunit: mRNA binding channel
-recognition of prokaryotic RBS
-decoding centre(charged tRNAs read/decode the codon units of mRNA)
-part of tRNA binding sites
-(interacts with anticodon loop)
-mRNA translocation
large subunit: peptidyltransferase centre (rRNA)(responsible for formation of peptide bond)
-nascent polypeptide exit channel
-part of the tRNA binding site
-(interacts with the acceptor arm)
-factor binding centre (GTPase associated region, GAR)
Describe the ribosome cycle
- translation begins with binding of the mRNA and initiating tRNA to a free, small subunit of the ribosome
- small subunit - mRNA inititator-tRNA complex then recruits large subunit to create intact ribosome with mRNA sandwiched btw 2 subunits
- protein synthesis initiated, commencing the start codon at the 5’ end of the message and progressing towards 3’ end of mRNA
- as ribosome translocates from codon to codon, 1 charged tRNA after another is slotted into the decoding and peptidyl transferase centres of ribosome
- when elongating ribosome encounters a stop codon, now completed polypeptide chain released and ribosome dissociates from the mRNA as separate large and small subunits
what 3 events needs to occur for translocation initiation
- ribosome recruited to mRNA
- charged tRNA must be placed into p site of ribosome
- ribosome must be precisely positioned over start codon
what are 3 binding site to perform peptidyl transferase rxn
A-binding site for amino acylated tRNA
P-binding site for peptidyl tRNA
E-binding site for tRNA that is released after the growing polypeptide chain has been transformed to aminoacyl-tRNA
describe mRNA binding path
mRNA wraps around neck of 30S subunit
what is degeneracy
when one amino acid can potentially have more than one genetic code
describe 4 features of genetic codes
- AUG is the most common translation initiation codon
- 2 translation termination codons
- mutation in the 3rd position is less likely to cause a change in amino acid at the protein level
- mutation in the 1st codon will give a similar amino acid in many cases
what is third base wobble
there are 20 standard amino acids. These 20 standard amino acids are encoded by 61 codons, each codon containing 3 bases. If you look closely at a list of codons that encode for a single amino acid, you may notice that one amino acid is encoded by many codons, usually with the first 2 bases the same, and the third codon variable. This third codon is known as a wobble, and the bond formed between this third base on the tRNA and the ribosome is not as stable, thus its importance is diminished.
G C A U I
U or C G U A or G A, U, or C
which base is more flexible
34 compared to 35, and 36
function of IF1, IF2, IF3
IF1-prevents tRNAs from binding to portion of small subunit that will become part of A site
IF2-GTPase (protein that binds and hydrolyzes GTP) that interacts with key components of initiation machinery (small subunit, IF1, and charged initiator tRNA(fMet-tRNAi)–facilitates the association of charged initiator with small subunit and prevents other charged tRNAs from associating with small subunit
If3-binds to small subunit and prevents it from reassociating with large subunit
Prokaryotic Translation Initiation Process
- IF3 binds to 30S subunit
- prevent the association of the 50S subunit with the 30S subunit
- binds to the E site in the 30S subunit - Binding od IF1
- blocks the A site
- recruits IF2-GTP - Binding of IF2-GTP
- binds close to P site - Recruitment of fMet-tRNAi and mRNA
- can be in any order
- the initiation codon is in P site
- fMet-tRNAi inserted at the P site by IF2
- IF3 binds to the G-C region in the anti-codon
- formation of 30S initiation complex - Anticodon in fMet-tRNAi base pairs with the initiation codon
- conformational change in 30S subunit
- dissociation of IF3 - Formation of the 70S initiation complex
- Binding of the 50S subunit
- stimulation of GTPase activity in IF2-GTP
- GTP hydrolyzed
- dissociation of IF2-GDP and IF1
- site is empty
- ready to accept a charged tRNA
Key steps in bacterial translation elongation
- Aminoacyl-tRNA binding
- mediated by EF-Tu-GTP
- GTP hydrolysis of EF-Tu-GTP
- Locking step*
- accomodation* - Peptide bond formation
- Translocation
How is charged tRNA delivered to A(T) site
EF-Tu-GTP binds to a charged tRNA
-protects the activated amino acid from hydrolysis
-delivers the charged tRNA to the assembled ribosomes (70S not 30S)
-ensures the correct codon-anticodon interaction
With proper codon-anticodon interaction
-EF-Tu interacts with the factor binding centre in the large subunit
-GTP hydrolysis
-Release of EF-Tu-GDP
Reloading of GTP to EF-Tu-GDP by EF-Ts
Possible interactions between codons and anticodons: selection by EF-Tu-GTP
12, 123,23* , 13
Possible interactions between codons and anticodons: selection by locking
12, 123,23
Two other mechanisms for selection of correct aminoacyl-tRNA
- Locking
- checking the correct pairing of the 1st two nucleotides in the codon with the anticodon by several conserved nucleotides (A 1492, A1493 and G530) in 16S rRNA - Correct paring of the 1st 2 nucleotides in codon-anticodon region
- allows hydrogen bond formation btw the 2 A nucleotides in 16S rRNA and the codon-anticodon structure in the A-site of the ribosome
- locks the mRNA-tRNA complex to the 30S subunit (16s rRNA)
