RNA is composed of _____ diff ntides, wh creates ____ possible combinations of 3 ntides (one codon). H/e, only ____ diff AAs are commonly found in proteins, and ea codon specifices ____ AA. Therefore, the genetic code is _______ and ________.
RNA is composed of four diff ntides, wh creates 64 possible combinations of 3 ntides (one codon). H/e, only 20 diff AAs are commonly found in proteins, and ea codon specifices only one AA. Therefore, the genetic code is redundant/degenerate and unambiguous.
- unambiguous - ea codon species only one AA
- degenerate - one AA may be specified by > 1 codon
- typ vary in third position/ntide; "wobble"
T/F: the genetic code is universal.
The genetic code is nearly universal.
- Slight diffs occur, but chiefly in mRNA of mitochondria and some fungi/protozoa.
What establishes the "reading frame" in mRNA translation?
A special signal at beginning of ea mRNA sets correct reading frame; like a punctuation mark.
- In principle, an mRNA seq can be translated in any one of three diff reading frames, dep on whr decoding process begins, but only one of wh specifies the correct protein.
Codons in mRNA do not directly recog the AAs they specify. Instead, xl deps on _________, wh recog/bind codons at one site and AA at another.
Codons in mRNA do not directly recog the AAs they specify. Instead, xl deps on tRNA "adapters", wh recog/bind codons at one site and AA at another.
RNAs typ fold into 3D struc via bp's b/w diff regions. How might this influence the structure of tRNAs?
RNAs typ fold into 3D struc via bp's b/w diff regions → if bp'd regions are strongly linked, struc folds back on itself to form double-helical struc (like DNA); tRNAs adopt similar struc.
- Four short segments of folded tRNA are double-helical → cloverleaf struc when drawn schematically.
- Cloverleaf struc undergoes further folding to form compact, L-shape; held t/g by additional H bonds b/w diff regions.
- Anticodon region at one end; AA attachment site at other.
tRNAs have two regions of unpaired ntides situated at either end of its compact L-shaped structure. Describe ea of this critical regions.
tRNAs have two regions of unpaired ntides situated at either end of its compact L-shaped structure:
Anticodon - set of 3 consec ntides that bp to complem codon in mRNA.
- anticodon bp w mRNA is antiparallel.
- AA attachment - Short, single-stranded region at 3′ end whr matching AA (complem to codon) is covalently attached to tRNA.
T/F: AAs are attached to tRNA via a covalent bond.
AA attachment site - Short, single-stranded region at tRNA's 3′ end whr matching AA (complem to codon) is covalently attached to tRNA.
- The energy stored in this bond is later used to drive xfr of polypeptide chain fr peptidyl-tRNA to aminoacyl-tRNA (via peptidyl transferase in large ribo subunit)
The genetic code is unambiguous and degenerate. What does this imply wrt tRNAs attaching AAs?
Genetic code is unambiguous and degenerate → some AAs have > 1 tRNA; some req accurate bp only at first two ntides of codon → can tolerate mismatch (or wobble) at third position.
- ~500 diff tRNA genes in humans; only 48 anticodons.
tRNAs are linked ("charged") w corresponding AA via ___________ (enzyme).
tRNAs are linked ("charged") w corresponding AA via aminoacyl-tRNA synthetases.
- Recall: AAs are covalently attached to single-stranded 3' end of tRNA.
T/F: there is a diff aminoacyl-tRNA synthetase for ea AA.
Diff synthetase enzyme for ea AA → 20 synthetases (bc 20 AAs).
- I.e. one synthetase attaches glycine to all tRNAs that recog codons for glycine.
- Ea synthetase recogs specific ntides in both anticodon and AA-accepting arm of tRNA.
Briefly describe the rxn catalyzed by aminoacyl-tRNA synthetase.
aminoacyl-tRNA synthetase covalently couples a partic AA to its corresponding tRNA(s) in a process called charging.
Coupled to (driven by) ATP hydrolysis:
- ATP → AMP + 2 Pi.
Produces high-energy bond b/w tRNA (3' end) and AA → bond energy later used to covalently link AA to growing polypep chain.
- Recall: charged tRNA notated as tRNAAA.
_________ are large complexes made fr dozens of small proteins and several crucial RNAs.
ribosomes are large complexes made fr dozens of small proteins (ribosomal proteins) and several crucial RNAs (ribosomal RNAs; rRNAs).
- A typ euk cell contains millions of ribosomes
Compare the structure of euk/prok ribosomes.
Euk vs prok ribosomes:
- v similar in struc/func: both composed of one large and one small subunit.
- Mass of several million daltons; compared to mass of average-sized protein of ~30,000 daltons.
- Euk ribosomes/subunits are larger: 40S + 60S = 80S vs 30S + 50S = 70S for proks.
What are the names of the three binding sites for tRNAs on ribosomes, and wh subunits are involved in the formation of these sites?
Ea ribosome contains three binding sites for tRNAs: A, P, and E sites:
- A = aminoacyl-tRNA
- P = peptidyl-tRNA
- E = exit
Only two of the three sites are occupied at any one time.
Both subunits are involved in forming APE sites, but only small subunit involved in mRNA binding.
T/F: Both ribosomal subunits are involved in forming APE (tRNA attachment) sites, but only the small subunit is involved in mRNA binding.
Both ribosomal subunits are involved in forming APE (tRNA attachment) sites, but only the small subunit is involved in mRNA binding
In the process of translation, a(n) (charged/uncharged) tRNA enters (A/P/E) site and (covalently bonds/base pairs) w complem codon on mRNA → new AA is linked to the peptide chain held by tRNA in neighboring (A/P/E) site → (large/small) subunit shifts forward → (charged/uncharged) tRNA moves into (A/P/E) site and is ejected.
In the process of translation, a charged tRNA enters A site and base pairs w complem codon on mRNA → new AA is linked to the peptide chain held by tRNA in neighboring P site → large subunit shifts forward → uncharged tRNA moves into E site and is ejected.
During translation, the polypeptide chain grows fr its ______ end (__-terminal) to its _______ end (__-terminal).
During translation, the polypeptide chain grows fr its amino end (N-terminal) to its carboxyl end (C-terminal).
Describe the steps of translation.
Steps of translation:
- Charged tRNA bp's to complementary codon (mRNA) in vacant A site.
- A and P sites are sufficiently close t/g → tRNAs are forced to bp w contiguous codons, i.e. no stray bases in b/w → preserves reading frame thru/o xl.
- Peptidyl transferase (in large ribo subunit) catalyzes the breaking of the covalent bond b/w carboxyl end of growing polyp chain attached to peptidyl-tRNA → forms peptide bond w free amino group of AA covalently attached to aminoacyl-tRNA.
- Large subunit translocates relative to small subunit → moves tRNAs in A/P sites into P/E sites, resp.
- Small subunit translocates exactly 3 ntides (one codon) along mRNA → restores position relative to large subunit, ejects uncharged tRNA fr E site, and resets A site to receive next charged tRNA.
At the start of euk translation, ________ is loaded in the __ site of the _____ ribosomal subunit, along w additional proteins called ____________.
At the start of euk translation, initiator tRNA (Met-tRNAi) is loaded in the P site of the small ribosomal subunit, along w additional proteins called euk initiation factors (eIFs).
- Met-tRNAi is distinct fr tRNA that normally carries Met (Met-tRNA), i.e. only a charged initiator tRNA is capable of binding tightly to P site in absence of large ribosomal subunit.
- Note that this occurs before small ribo subunit binds mRNA and w/o large ribo subunit.
How is initiator tRNA different fr the tRNA that normally carries Met?
Met-tRNAi is distinct fr tRNA that normally carries Met (Met-tRNA), i.e. only a charged initiator tRNA is capable of binding tightly to P site in absence of large ribosomal subunit.
- Recall: Met-tRNAi binds to P site (not A site), and only the small subunit is present during xl initiation.
Euk translation begins w Met-tRNAi binding the P site of the small ribo subunit, along w additional euk initiation factors (eIFs; proteins). Describe the following steps leading up to the first peptide bond.
Met-tRNAi binds P sites of small subunit → eIFs bind → ...
- Small subunit binds to 5' end (cap) of mRNA.
- Small subunit moves along mRNA until it recogs start codon (AUG).
- Movement powered by IFs that act as ATP-powered helicases.
- Several IFs dissoc fr small subunit to make way for large subunit → completes ribosomal assembly.
- Protein synth (4-step cycle) begins w addition of next charged tRNA to A site.
- Recall: Met-tRNAi already in P site.
- Recall: 4-step cycle: carboxyl end of AA in P site is uncoupled fr tRNA → peptide bond formed w free amino gr of AA attached to tRNA in A site → large subunit translocates → small subunit translocates to next codon and ejects uncharged tRNA fr E site → repeat.
Bacterial mRNAs have no 5' cap. How, then, is translation initiated in proks?
bac mRNAs have no 5′ cap → instead have specific ribosome-binding seqs (Shine-Dalgarno seqs) located a few ntides upstream of AUGs at wh xl is to begin.
- Unlike euk ribosomes, prok ribosomes can readily bind directly to a start codon that lies in the interior of an mRNA, as long as a ribosome-binding site precedes it by several ntides.
- Necessary bc prok mRNAs are often polycistronic, i.e. encode several diff proteins, ea wh is translated fr same mRNA.
In the process of translation termination, __________ bind to a stop codon in __ site → causes ____________ to catalyze addition of ____ to peptidyl-tRNA → ______ end of growing chain is released fr tRNA → polypeptide released.
In the process of translation termination, release factors (proteins) bind to a stop codon in A site → causes peptidyl transferase to catalyze addition of water to peptidyl-tRNA → carboxyl end of growing chain is released fr tRNA → polypeptide released.
- Ribosome releases mRNA → dissociates into its subunits.
Ribosomes are composed of a large and small subunit. What primary role does ea subunit serve?
Small subunit: matches tRNAs to codons of mRNA.
Large subunit: catalyzes formation of peptide bonds.
Ribosomes are considered "ribozymes". What does this name imply about its structure and function.
The ribosome is a ribozyme → rRNAs—not protein subunits—are resp for overall 3D struc and ability to catalyze protein synth.
- Ribosomal proteins are typ located on surface → fill gaps/crevices of the folded RNA.
rRNAs—not protein subunits—are resp for the overall 3D struc and ability of ribosomes to catalyze protein synth; hence, "ribozymes". As such, what role do ribosomal proteins serve?
Ribosomal proteins are typ located on surface → fill gaps/crevices of the folded RNA.
- Main role: help fold/stabilize RNA core while permitting changes in rRNA struc that are necessary for efficient protein synth.
Where are the tRNA binding sites and peptide formation catalytic site located in/on ribosomes?
rRNAs are folded into highly compact, precise 3D strucs wh form core of ribosomes:
- Form the tRNA binding sites (APE)
Catalytic site—peptidyl transferase (peptide bond formation)—is formed by the 23S rRNA of the large subunit.
- Peptidyl transferase is a highly structured pocket → precisely orients two reactants (growing polypeptide and charged tRNA) → greatly ↑ probability of rxn.
T/F: Specific codons in mRNA signal the ribosome where to start/stop protein synth.
Specific codons in mRNA signal the ribosome where to start/stop protein synth.
- In vitro, ribosomes can be forced to translate any RNA.
- In vivo, a specific start signal is req'd to initiate translation.
- Recall: start site sets reading frame.
New proteins all have ___ as its first AA at the __-terminus; typ removed after xl by a ________.
New proteins all have Met as its first AA at the N-terminus; typ removed after xl by a protease.
- Specified by codon 5'-AUG-3' → anticodon 3'-TAC-5'
- Bac: modified form of Met, formyl-methionine.
T/F: In both proks/euks, translation termination is signaled by stop codons (UAA, UAG, UGA), wh are not recognized by any tRNAs.
In both proks/euks, translation termination is signaled by stop codons (UAA, UAG, UGA), wh are not recognized by any tRNAs.
- I.e. no AA specified by stop codons.