Biochem 4.5 - RNA Translation

In terms of their sedimentation coefficients, the 80S eukaryotic ribosome consists of:

(1) 60S large subunit (2) 40S small subunit

In addition to binding sites for mRNA transcript and the nascent polypeptide chain, the ribosome harbors three distinct sites for simultaneously accommodating three tRNAs at various stages:

(1) A-site for aminoacyl-tRNA (2) P-site for peptidyl-tRNA (3) E-site for deacylated tRNA

RNA translation into the synthesis of a polypeptide chain within the ribosome can be divided into three major stages:

(1) Initiation (2) Elongation (3) Termination

(1) A-site for aminoacyl-tRNA

(the incoming tRNA carrying an *Amino acid)

(3) E-site for deacylated tRNA

(the tRNA about to *Exit after donating its amino acid)

(2) P-site for peptidyl-tRNA

(the tRNA attached to the nascent *Polypeptide)

Translation Elongation: Overview

1) Decoding 2)Transpeptidation 3) Translocation

Eight Stages for Translation Initiation

1) Met-tRNAi^Met Priming (2) Met-tRNAiMet Transport (3) mRNA Packaging (4) mRNA Attachment (5) mRNA Scanning (6) Initiation Codon Recognition (7) Ribosomal Subunit Association (8) Initiation Complex Formatio

The task of removing the aminoacyl group of aminoacyl-tRNA and adding it to a growing polypeptide chain falls to an RNA component (__S) of __S subunit called "______ _________"

28S; 60S subunit peptidyl transferase

On the other hand, the _____ of the acceptor stem of tRNA covalently attaches via an ____ linkage to one of the 20 amino acids in a highly ____ manner

3'-OH end ester specific

Thus, the actual number of tRNAs required for productive protein synthesis is much less than 61—but a minimum of ___ tRNAs are required to recognize all 61 mRNA codons according to "______ ________"

32 Wobble hypothesis

Prior to the formation of PIC, the separated __S subunit of ribosome recruits ____, ____ and ____ (and presumably ____)

40S eIF-1 eIF-1A eIF-3 eIF-5

Transportation of GTP-eIF2-metRNAi^Met complex to the ribosome so as to promote the formation of the so-called __S complex — or ________ (___) — requires a pool of separated 40S and 60S ribosomal subunits

43S complex Preinitiation complex (PIC)

Ribosome reads the mRNA transcript in the ______ direction.

5' --> 3'

The _____ of mRNAs usually possesses a stem-loop secondary structure that can impede their loading onto the ribosomes

5'-UTR

Although mRNAs harbor multiple AUG codons, the first (or the leading) AUG codon immediately following the _____ sequence serves as the initiation codon or ________ (_____)

5'-UTR translation initiation site (TIS)

In sync with the assembly of the p48S complex, the 40S ribosome begins to scan the mRNA transcript downstream of the _____ to locate the AUG initiation codon.

5'-cap

The release of eIFs from the 48S initiation complex makes way for the docking of the __S subunit—which binds to the same interface on 40S subunit that had earlier "piggybacked" the eIFs

60S subunit

Of the 64 codons, only __ encode amino acids— the other three serve as "_____" or "____" codons in that they do not specify an amino acid but rather signal the end of the polypeptide chain when encountered by the ______ translational machinery

61 nonsense or stop ribosomal

Since mRNA is comprised of only four nucleotides (A/C/G/U), there is a total of __ three-letter genetic codons—ie there is a total of 64 possible variants made up from 4 distinct letters arranged in 3 different ways (4^3 = 64)

64

(1) eIF(4E) binds to the ______________ (___) @ the 5'-cap of mRNA

7-methylguanosine (m7G)

1S = ? SI Units

= 10^-13 s (or 100 fs)

The 80S complex is fully "primed" to accept/recruit an incoming aminoacyl-tRNA at the____ in order to "stitch" its aminoacyl group onto the ___-terminal of methionine within the _______ waiting @ the ____.

A-site C methionyl-tRNAi^Met P-site

(1) The newly synthesized peptidyl-tRNA @ the __-site must be translocated to the __-site

A-site P-site

In this manner, the ribosome selects and recruits an aa-tRNA whose anticodon is complementary to the mRNA codon @ the __-site in a phenomenon referred to as "______"

A-site decoding

eEF(1A) and eEF(1B) specifically bind to tRNAs only ___ they become aminoacylated—ie ______

AFTER aa-tRNAs

On the other hand, the ___ codon serves as "_________ (___)" for the synthesis of a polypeptide chain

AUG (Met) translation initiation site (TIS)

(5) translocation of uncharged/deacylated tRNA from P-site to ______.

E-site

eRF(3) undergoes _____ exchange in order to induce a ______ change within the ribosome so as to facilitate the release of eRF(1)

GDP-GTP conformational

Upon the cleavage of the ester bond, ________ loads onto the ribosome to accelerate the release of the cleaved polypeptide

GDP-bound eRF3

Coupled with ___ hydrolysis, the incoming aminoacyl-tRNA (aa-tRNA) is escorted/delivered by ____ and ____ to the A-site within the ribosome

GTP eEF(1A) eEF(1B)

Binding of _________ to the 48S complex triggers eIF2-~catalyzed hydrolysis~ of pre-bound GTP to ___ —an event that results in the release of all eIFs but _____.

GTP-bound eIF(5B) GDP eIF(1A)

Rather, many tRNAs can base pair with more than one mRNA codon—eg tRNA^Ala harboring the ____ anticodon recognizes three (GCA/GCC/GCU) of the four mRNA codons for ______ (the fourth codon being GCG)

IGC alanine

The AUG initiation codon is recognized by the so-called initiator tRNA covalently attached to its cognate ____ residue (___________) — virtually all eukaryotic proteins begin with Met residue though this is sometimes cleaved off during post-translational modification (PTM).

Met methionyl-RNAiMet

Polypeptide synthesis proceeds from the __ to __ terminus.

N- C-

The GTP-eIF2-metRNAi^Met complex subsequently translocates to the _____ within the __S subunit of ribosome so as to transport the metRNAi^Met in order to allow it to base pair (via its ____ anticodon) with the AUG initiation codon within ____.

P-site 40 CAU mRNA

Within the 43S complex, methionyl-tRNAi^Met binds to the ____ so as to essentially serve as a __________—thus being in a position to receive the second amino acid from the next aa- tRNA as dictated by the mRNA codon following the initiating AUG codon

P-site proto-peptidyl-tRNA

___ ___—an RNA component of ribosome (ribozyme)—catalyzes the formation of a ____ bond through the nucleophilic displacement of the ___- tRNA @ the P-site by the _____ group of aa-tRNA @ the A-site in a reaction referred to as "______"

Peptidyl transferase peptide peptidyl aminoacyl transpeptidation

Upon the release of eRF1, ___ loads on to the ribosome and, in so doing, displaces GTP- bound eRF3

Rli

The AUG initiation codon (or____) of mRNA is embedded within the GCC(A/G)CCAUGG consensus sequence

TIS

What is the difference between tRNA^Arg and arginyl-tRNA?

The tRNA^Arg is the uncharged (free) tRNA that has the capacity to bind Arg, whereas arginyl-tRNA is the tRNA^Arg in which Arg is covalently attached to its 3'- OH group—strictly, Arginyl-tRNA should be written as "arginyl- tRNA^Arg"

When the ribosome encounters one of the three stop codons—___, ___ or ___—at the ___-site, it comes to a grinding halt due to the fact there is no aa-tRNA counterpart that can base pair with one of these stop codons in mRNA

UAA UGA UAG A-site

There exists at least one tRNA for each amino acid—and so does at least one specific ____ (eg arginyl-tRNA synthetase and so forth) — not only that but many "abundant" amino acids have more than one _____/_____ pairs.

aaRS cognate tRNA / aaRS

In order to decode the three-letter genetic code within a mRNA into a polypeptide chain, an ______ is required—this role is delegated to the so- called "________"

adaptor transfer RNA (tRNA)

Simply put, the ability of anticodon to somewhat "wobble" at the third position of the mRNA codons accounts for its ability to recognize multiple mRNA codons specifying the same ______ in what has come to be called the "______ ________"

amino acid Wobble hypothesis

(1) Activation of a specific amino acid (aa) with ATP to generate the "high-energy" _______ (____) intermediate: aa + ATP <=> (____) + PPi

aminoacyl-adenylate (aa-AMP) aa-AMP

How is the synthesis of aa-tRNA directed to ensure that only the correct amino acid attaches to a specific tRNA harboring its anticodon?

aminoacyl-tRNA synthetase (aaRS)

Synthesis of aminoacyl-tRNA (aa-tRNA) is driven by ___________ (___) via two steps.

aminoacyl-tRNA synthetase (aaRS)

In this manner, aaRS-catalyzed synthesis of aa-tRNA ensures that it harbors the correct ______ for the ________ it is carrying— aaRS thus serves as a "______" in that it matches a specific amino acid with its anticodon within the cognate tRNA.

anticodon amino acid matchmaker

sedimentation coefficient (c)

c = m/6πnr [1] m = mass of the particle r = radius of the particle (a measure of its volume/surface area) n = viscosity of the solution

Peptidyl transferase promotes nucleophilic attack of the incoming amino group of aminoacyl-tRNA (A-site) on the ____ group of peptidyl-RNA (P-site)

carbonyl

In order to serve as the link between DNA and proteins, mRNA harbors a "____" in the form of its _____ sequence

code nucleotide

Since a polypeptide chain is comprised of 20 amino acids, each residue can be specified by more than one ____ (except for ___ and ___)—ie the genetic code is _____.

codon Met (AUG) Trp degenerate

After the dissociation of deacylated tRNA from the E-site, the above reaction is repeated for the next aminoacyl-tRNA as dictated by the next ____ in the _____ sequence.

codon mRNA

Each aa-tRNA conjugate enables the amino acid to be added to a growing polypeptide chain within the ribosomal translational machinery in an ordered manner solely on the basis of __________ ______ _______ ________ —the aminoacyl group of aa-tRNA has NO say on this "match-making" process.

codon- anticodon complementary base pairing

(2) The aa-AMP intermediate subsequently reacts with a _____ ______ (ie one that harbors the anticodon for the specific aa to be covalently attached): aa-AMP + tRNA <=> aa-tRNA + AMP

cognate tRNA

Since c depends on both the mass and surface area, the sedimentation coefficients of two particles measured separately do NOT add up to the value obtained together as a ______.

complex

(2) 40S small subunit

comprised of a single RNA polynucleotide (18S) and ~33 polypeptides

(1) 60S large subunit

comprised of three RNA polynucleotides (28S/5.8S/5S) and ~49 polypeptides (proteins)

Such codon-anticodon base pairing induces a _______ change within the p48S complex to generate the 48S complex

conformational

Working in sync with each other, the eIFs within the 43S complex induce a ________ change within the 40S ribosomal subunit so as to accommodate the pre-packaged mRNA ______.

conformational transcript

Notably, non-Watson-Crick base pairing between tRNA and mRNA allows a greater degree of ________ ________ (at the third position of the codon) needed to accommodate the same tRNA within degenerate codons—such non-canonical pairing is referred to as "______ ____ ______"

conformational freedom Wobble base pairing

Such a code (or information) is divided up into a _____ triplet of bases (or words) without any punctuations

continuous

(2) The uncharged or _____- tRNA already occupying the P-site must be transferred to __-site—

deacylated E-site

In order to "translate" the genetic information (or genetic code) carried by mRNA into the synthesis of a polypeptide chain, it must first be ______.

decoded

mRNA codons do not ____ interact with their corresponding amino acids

directly

The 40S-eIFs complex subsequently serves as a _____ _____ for the recruitment of GTP-eIF(2)- metRNAi^Met complex resulting in the formation of _________ (or PIC)

docking platform 43S complex

Such translocation of various components is facilitated by the binding of ___ to the ribosome in a GTP-dependent manner—and the subsequent hydrolysis of GTP returns the ribosome to "____ ____" of its elongation cycle

eEF2 ground zero

In particular, the eIF(4E)-eIF(4G)-eIF(4A) (collectively called _____) ______ complex plays a central role in the recognition of not only TIS at the 5'-end but also the ______ at the _____ of mRNA:

eIF(4F) heterotrimeric poly(A) tail 3'-end

This event releases both ___ and ____ from the p80S complex coupled with a _______ change that ultimately generates the 80S complex

eIF(5b) eIF(1A) conformational

The ability of methionyl-tRNAi^Met to recognize the AUG initiation codon arises from its ability to specifically bind to ___ and the _____ within the __S subunit of ribosome—on the other hand, methionyl-tRNA^Met that recognizes AUG -internal- codons can only be accommodated within the A- site thus barring it from initiating protein synthesis

eIF2 P-site 40

Association of 60S subunit with 40S subunit within the p80S complex triggers ____-catalyzed hydrolysis of its bound GTP to GDP

eIF5B

Such mRNA scanning requires the cooperation of all parties including, in particular, the attached ____, albeit to varying degrees —eg omission of ____ mitigates the scanning process, while the lack of ____ completely halts/abrogates it

eIFs eIF(1A) eIF(1)

Upon recognition and confirmation of one of the stop codons at the A-site within the ribosome, ____ triggers the hydrolysis of the ___ bond in peptidyl- tRNA @ the P-site—thereby releasing the newly synthesized polypeptide

eRF(1) ester

The release of free energy upon the cleavage of "high- energy" ____ linkage in peptidyl-tRNA drives the reaction to completion.

ester

The covalent attachment or ________ of a tRNA with a specific amino acid (aa) for which it harbors an anticodon generates the corresponding _________ (____) conjugate in a process referred to as "______"

esterification amino acyl-tRNA (aa-tRNA) aminoacylation

At this stage, the ribosome releases eIFs and gets ____ (___) to begin the next stage of translation _____.

eukaryotic elongation factors (eEFs) elongation

Eukaryotic translation initiation also requires a plethora of helper proteins called "___________ (_____)" and can be divided into eight stages.

eukaryotic initiation factors (eIFs)

The AUG initiation codon, or TIS, is embedded within the GCC(A/G)CCAUGG consensus sequence—a motif that is recognized by the binding of various helper proteins called "________ (____)"

eukaryotic initiation factors (eIFs)

Such halting signals the loading of _____ (____) such as ____, ____ and ____ —also called _______

eukaryotic release factors (eRFs) eRF(1) eRF(3) Rli1 ABCE1

Such 3D shape is stabilized by _____ _______ interactions as well as ____________ base pairing between bases in various stems

extensive stacking non-Watson-Crick

Collectively, the 64 codons constitute what has come to be known as the "_____ ____"—ie a language made up of only 4-letter alphabet, 3-letter words, and 64 words in total

genetic code

eIF(4A) is a DEAD-box RNA _____ that promotes the unwinding of 5'-UTR (in an ATP-dependent manner)—in the presence of _____ —to prepare the mRNA transcript for subsequent ribosomal attachment in the form of ___________.

helicase eIF(4B) mRNA-eIFs complex

In an ultracentrifuge, larger particles (of equal density) sediment faster and thus have a ______ c —the higher the value of c the ____ the particle and vice versa

higher larger

The various secondary structural motifs constituting the 2D cloverleaf form of tRNA fold into a complex 3D structure resembling the _______

letter L

Next, Rli facilitates the release of bound ____ and _____ from the ribosome

mRNA deacylated tRNA

tRNA is the missing link between _____ and _____ synthesis

mRNA protein

Each aa-tRNA conjugate enables amino acid to be added to a growing polypeptide chain within the ribosomal translational machinery in an ordered manner as specified by the _____ _____.

mRNA codon

This step thus completes the unification of ___ ______ with ________ under the watchful eye of the 40S ribosomal subunit

mRNA transcript metRNAi^Met

After the preparation of mRNA with various eIFs, the ____ ____ is shuttled to the ribosome, where it attaches to the ____ ______ pre-loaded with metRNAi^Met to form the pre-___S (p__S) complex

mRNA-eIFs complex 43S complex pre-48S (p48S) complex

c is dependent on both the ____ and ________ of particles — it is indicative of how quickly they settle (or sediment) to the bottom of a liquid (in a tube) when it is spun down in an __________.

mass surface area ultracentrifuge

However, both the initiator methionyl-tRNAi^Met and internal methionyl-tRN^Met are aminoacylated with Met residue by the same _______ (____)

methionyl-tRNA synthetase (MetRS)

While the eIFs play a central role in guiding the 40S ribosomal unit to the initiation codon, its ultimate recognition is dependent upon the ability of ______ to base pair (via its ___ anticodon) with the AUG codon within mRNA.

methionyl-tRNAi^Met CAU

Such nucleotide modification imparts upon tRNAs the ability to base pair with more than one mRNA codon by virtue of its ability to engage in ______________ base pairing at the _____ position

non-Watson-Crick third

Paradoxically, the genomes of most eukaryotes encode over 100 tRNAs—many of these tRNAs encode _______ amino acids while others are _______ or represent _________.

non-standard non-functional pseudo-genes

For each of the 20 amino acids, there exists at least ___ cognate tRNA harboring an anticodon to match the corresponding codon in ______.

one mRNA

Each codon encodes ____ of the 20 amino acids in a polypeptide chain as well as a ____ signal and a ____ signal

one start stop

(1) Displacement of the tRNA of the ______-tRNA (harboring n amino acids)

peptidyl

This reaction thus not only increases the peptide length of the ____-tRNA from n to n+1 but also moves it to the A-site.

peptidyl

(2) eIF(4G) binds to ____________, thereby _____ the mRNA— presumably to "tether" eIF(4F) complex to the poly(A) tail so that it can remain bound to the mRNA for subsequent rounds of translation rather than falling off—thereby enhancing its processivity.

poly(A)-binding (PAB) protein circularizing

Multiple ribosomes can bind to a single mRNA transcript adopting a "beads- on-a-string" appearance called "______" or "_______".

polyribosome polysome

On the other hand, tRNA cognates for corresponding amino acids are usually _______ with the three-letter amino acid code.

post-superscripted

The association of 60S subunit with the 48S complex generates the pre-__S (p__S) complex

pre-80S (p80S)

Ribosome undergoes a constant recycling process whereby it assembles ___ to initiation of each round of polypeptide synthesis (that terminates with the dissociation of ribosomal subunits)

prior

(2) Elongation

requires eukaryotic elongation factors (eEFs)

(1) Initiation

requires eukaryotic initiation factors (eIFs)

(3) Termination

requires eukaryotic release factors (eRFs)

In so doing, tRNAs transfer or transport their cognate amino acids to a growing polypeptide chain guided by the ______ translational machinery

ribosomal

The ribosome is made up of two constituent subunits (small and large) — each subunit is a complex set of ______ (____) and ______.

ribosomal RNAs (rRNAs) proteins

Translational machinery (or ______) is the site where the genetic code within the mRNA is "decoded" or "translated" into a polypeptide chain (protein).

ribosome

Peptidyl transferase is a ______ —an exclusively RNA-based catalyst (or a _____ ________ _______)

ribozyme ribonucleic acid enzyme

The various components of the ribosome are described in terms of their _____________ (_) — expressed in the non-SI units of _______ (_).

sedimentation coefficient (c) Svedberg (S)

Although there are 61 functional mRNA codons (encoding only 20 amino acids), recognition of each mRNA codon does not necessarily require a unique ____ —ie 61 cognate tRNAs

tRNA

Rather, a specific ____ —carrying a cognate amino acid— base pairs via its _____ with a corresponding mRNA codon

tRNA anticodon

How is the genetic code in the form of a contiguous trinucleotide sequence within mRNA decoded into amino acids vis-à-vis the synthesis of a polypeptide chain?

tRNA ...

In order to recognize the AUG initiation codon, metRNAi^Met is first assembled into a ______ complex with ____________ so as to prepare or "prime" it for transport to the ribosome

ternary GTP-bound eIF2

The site of mRNA codon degeneracy at the ____ position is exquisitely mirrored by anticodons in tRNAs in that the latter harbor a modified base such as _______ at the complementary position— ***ie the 5'-nucleotide in tRNA anticodon that base pairs with the 3'-nucleotide (the third position) in mRNA codon

third inosine (I)

Multiple codons specifying a unique amino acid usually only differ at their ____ base (the site of codon degeneracy)—such "_______" nature of the genetic code is evidence that it must have evolved to minimize the ______ effects of mutations!

third nonrandom deleterious

tRNA is a polymer (typically 70-90nt long) comprised of ____ stem-loops and a solo stem called the "____ ____"— such structural motifs impart upon tRNA the so-called "____" form @ ____ structural level

three acceptor stem cloverleaf secondary

In order to prepare the ribosome for the next elongation cycle—[ie to add the next aminoacyl group of aa-tRNA to the nascent (growing) polypeptide chain as dictated by the codon-anticodon complementarity] —the following events that together constitute the "_____" phase have to be accomplished:

translocation

(2) Formation of a new peptide bond with the incoming amino acid (______)

transpeptidation

One of the three stem-loops within tRNA harbors a _____ sequence that is complimentary to one of the mRNA _____ —such a complimentary triplet of bases within tRNA is referred to as the "_____", and the corresponding stem-loop the "____ ___"

trinucleotide codons anticodon anticodon arm

Genetic code is a set of _____ of bases—each ____ is termed _____ — corresponding to the contiguous sequence of mRNA without any spaces or punctuations.

triplet triplet codon