ch.15: translation (part 1)

eIF2

(eukaryotes) a GTP-binding protein, escorts initiator tRNA to small subunits charged with methionine positions the Met-tRNA in the P-site of the initiator factor-bound small subunit.

eIF1

(eukaryotes) binds to E site while eIF1A, eIF3 and eIF5 bind to the small subunit.

IF1

(prokaryotes) prevent tRNAs from binding to the portion of the small subunit that will be apart of the A-site. (binds to the A-site)

IF3

(prokaryotes) binds to the small subunit and blocks it from reassociating with the large subunit (occupy the future E-site)

IF2

(prokaryotes) is a GTPase that interacts with three key components of the initiation machinery: the small subunit, IF1, and the charged tRNA. It facilitates the association of fMet-tRNA with the small subunit and prevents other charged tRNAs from associating with the small subunit

translation elongation

1. Correct aminoacyl tRNA loaded into A site of ribosome as dictated by codon 2. Peptide bond formed between aminoacyl-tRNA in A site and peptide chain that is attached to peptidyl-tRNA in P site. 3. Peptidyl-tRNA in the A-site and its associated codon must be translocated to the P=site so that the ribosome is ready for another cycle of recognition. EF-Tu-escorts aminoacyl-tRNA's to ribosome. Binds and hydrolyzes GTP. Can only bind to aminoacyl-tRNA when bound to GTP.

mechanisms for accuracy

1. Two adjacent adenine residues in 16S rRNA component of A site. Form H bonds with minor groove of each correct base pair formed between anticodon and first two bases of codon. 2. GTPase activity of EF-Tu. Release of EF-Tu requires GTP hydrolysis, which is highly sensitive to correct codon-anticodon base pairing. If wrong, alters ability of EF-Tu to hydrolyze GTP. Kinetic selectivity. 3. Proofreading when EF-Tu released. Charged tRNA first introduced into A site in a complex with EF-Tu-GTP, its 3' end is distant from the site of peptide bond formation. tRNA must rotate into peptidyl transferase center of large subunit-called accommodation. 3' end moves almost 70 angstroms, incorrectly paired tRNA's frequently dissociate from ribosome during this.

minor groove interactions

Additional hydrogen bonds are formed between 2 adenine residues of the 16S rRNA and minor groove of the anticodon-codon pair only when the first two bases of the anticodon-codon pair form correct watson-crick base pairs contribute to specificity of elongation

E, A

For a new round of peptide chain reaction to occur, the P-site tRNA must move to the ___ site and the ___ site tRNA must move to the P-site

IRES (internal ribosome entry site)

Function like prokaryotic RBS. Recruit small subunit to bind and initiate in absence of 5' cap Some do not need initiation factors-idea is that early mRNA had these and transcription factors evolved to make the process more versatile. MECHANISMS: recruit eIFG allow for bypass of initiation factors and initiation tRNAs

editing

Initial binding and adenylylation of amino acid then _____

poly-A tail

Modified end of the 3' end of an mRNA molecule consisting of the addition of some 50 to 250 adenine nucleotides. Added enzymatically and enhances level of translation. Also prevent degradation.

polycistronic mRNA

Multiple ORFs are called _______ often encode proteins that perform related functions

tRNA accommodation

Only correctly base-paired aminoacyl-tRNA remains with the ribosome as they rotate into the correct position for peptide-bond formation rotation = _______ During this, the 3' end of the aminoacylated tRNA moves almost 70A

factor-binding center

EF-Tu GTPase is active when it is associated with the _______, the same domain in the large subunit that activates the IF2 GTPase when the large subunit joins the initiation complex. EF-Tu only interacts with this after tRNA enters the A-site and a correct codon-anticodon match is made; At this point, EF-Tu hydrolyzes GTP and releases from ribosome.

large and small

Each tRNA binding site is formed at the interface between _________ subunits of the ribosome In this way the bound tRNA can span the distance between the peptidyl transferase center in the large subunit and the decoding center in the small subunit

2'-OH

Elimination of the ______ of a highly conserved residue in the 23S rRNA reduces the rates of catalysis by at least 10~ fold. often at end of P-site substrate-assisted catalysis

5' cap

Eukaryotic mRNA's recruit ribosomes using ______-recruits ribosome, once bound, ribosome moves in 5'->3' direction to find start codon. (process called scanning) also eukaryotic initiation factors

ORF (open reading frame)

Protein coding region of each mRNA is composed of contiguous, non-overlapping string of codons called the ______. Specifies a single protein and starts and ends at internal sites within mRNA. distant form ends of mRNA

codons

Protein coding region of mRNA consists of ordered series of 3 nucleotide long units called ______

blindly

Ribosome accepts tRNA's ______ so tRNA synthetases must be accurate.

variable loop

Site on the tRNA that accounts for different sizes; sits between anticodon loop and ѰU loop, varies between 3 and 21 bases

energy

Small _____ differences between isoleucine and valine pockets on tRNA; also size

mRNA

_______ binds to small subunit in anticodon region The 3' ends of the tRNAs that are coupled to the amino acid or to the growing peptide chain are adjacent to the large subunit. The anticodon loops of the bound tRNAs are located adjacent to the small subunits

50S, 30S

_______ contains 5S and 23S rRNA, where _____ has a single 16S rRNA

specificity

_______ determinants for aminoacyl-tRNA synthetases; clustered at two different sites on molecule. Acceptor stem and anticodon stem. Each amino acid specified by more than one codon, recognition of anticodon cannot be used

translocation

___________ initiated in large subunit first, then small subunit. tRNA's said to be in hybrid states. 3' ends shifted to new location but anticodon ends still in pre-peptidyl transfer positions. To complete this, second elongation factor called EF-G required.

ribosome

a macromolecular machine that directs the synthesis of proteins at least 3 RNA molecules, and more than 50 different proteins, speed: only 2-20 amino acid per second. In prokaryotes, the transcription machinery and the translation machinery are located in the same compartment. Thus this can commence translation of the mRNA as it emerges from the RNA Polymerase. In eukaryotes, transcription is in the nucleus, whereas translation is in the cytoplasm does little to prevent an incorrectly charged tRNA from adding an inappropriate amino acid to the growing polypeptide.

charged tRNA

a transfer RNA molecule to which the appropriate amino acid has been attached Requires acyl linkage between carboxyl group of amino acid and 2' or 3' hydroxyl group of adenosine nucleotide.

tRNAs

adaptors between codons and amino acids; 75-95 nucleotide sequences. Each end at 3' terminus with 5'-CCA-3', site attaches amino acid each is attached to a specific amino acid, and each recognizes a particular codon or codons unusual bases lead to improved tRNA function (pseudouridine (ψU), dihydrouridine (D), hypoxanthine)

EF-Tu

aminoacyl-tRNAs are delivered to the A-site by elongation factor ______ binds to tRNA 3' end once tRNA is aminoacylated, masking coupled amino acid can only bind when associated with GTP interacts with factor-binding center after tRNA enters the S site and correct codon-anticodon match is made . then it is released

tRNA synthetase

an enzyme that attaches the appropriate amino acid onto its tRNA required by tRNA charging can have up to 20 different ones; charing tRNAs for particular amino acids

class I enzymes

attach amino acid to 2'Oh of tRNA and monomeric needed in tRNA charging

class II enzymes

attach amino acid to 3'OH of tRNA and are dimeric or polymeric. needed in tRNA charging

A site

binding site for aminoacylated-tRNA; the growing peptide chain is linked to the tRNA here must move to P-site for a new round of peptide chain reaction to occur

eIF3

blocks premature association with 60S in eukaryotic ribosome big (as large as 40S subunit) and interacts with every member of the 43S PIC eIFG-mRNA interacts with this, forming the *48S preinitiation complex*

stop codon

codon that signals to ribosomes to stop translation UAA, UAG, UGA

genetic code

composed of only four nucleotides. Each codon that is read by a ribosome is composed of a distinct three-nucleotide sequence. There are sixty-four total codons that can be made using four different nucleotides, however, there are only twenty different amino acids created by ribosomes. What is the reason for this discrepancy? More than one codon sequence can code for the same amino acid.

pseudouridine(ѰU)

derived from uridine by isomerization in which uracil base switched from nitrogen 1 to carbon 5 (connection between base and ribose) helps improve tRNA function

dihydrouridine (D loop)

derived from uridine enzymatically Looks like cloverleaf

EF-T

does same thing as EF-G for EF-Tu

EF-G

elongation factor that completes process of translocation; binds with GTP after peptidyl reaction, EF-G-GTP binds and contacts factor binding center of large subunit, stimulates GTP hydrolysis. "Unlocks" ribosome. Gates separate E,P,A sites and the hydrolysis opens these gatees. EF-G-GDP binds to A site. Altered conformation stabilizes hybrid state of ribosome. Moves A site tRNA to P site, P site to E site, like dominoes. Accompanied by clockwise rotation of small subunit back to starting position. EF-G-GDP released and returns ribosome to locked state in which tRNA and mRNA associated with small subunit. has similar structure to tRNA

ATP, GTP, GTP

energy consumption in peptide formation: 1 ____ used by aminoacyl-tRNA synthetase _____ consumed in delivery of tRNA to A site of ribosome by EF-Tu 1 ____ used EF-G mediated process of translocation

kozak sequence

helps the ribosome identify the start codon in eukaryotes presence of purine three bases upstream of start codon and guanine immediately downstream. For example 5'G/ANNAUGG-3' presence increases translational efficiency.

editing pocket

how to ensure accuracy? Proofread products of charging reaction. isoleucyl-tRNA synthetase has nearby ______- that allows it to proofread product of adenylylation reaction. AMP valine can fit into pocked where it is hydrolyzed and released. AMP-isoleucine too large to fit into pocket and not subject to hydrolysis.

methionine

in eukaryotes, initiator tRNA is charged with ______

stabilize tRNA structure

in order to _______: 1. Formation of extended regions of base pairing results in base stacking interactions. 2. Hydrogen bonds are formed between bases in different helical region 3. Interactions between base and sugar-phosphate backbone

N-formyl methionine

in prokaryotes, initiator tRNA is charged with ________

anticodon loop

in tRNA structure, contains anticodon, 3 nucleotide long sequence. Recognizing basepairing

polyribosome (polysome)

mRNA that contains multiple ribosomes

at least

mRNAs contain ______ one ORF

decoding center

mRNAs must be threaded through the _______ during translation, and nascent polypeptide chain must escape from the peptidyl transferase center. in the small subunit these channels are only wide enough for unpaired RNA to pass through

eIF4G

mediates binding of translation initiation factors binds directly to the 3' end of the mRNA and the poly-A binding protein, results in the circular configuration of the mRNA and several rounds of translation

translational coupling

prokaryotic ORFs that lack a strong RBS, but are still translated, have ORFs that overlap translation of the downstream ORF requires translation of the upstream ORF

peptidyl transferase reaction

reaction to form a new peptide bond The aminoacyl-tRNA is attached at its 3' end to the carboxyl group of the amino acid bond between peptide-tRNA and growing peptide is broken (bond between aminoacyl-tRNA and amino acid is NOT BROKEN). this reaction takes place without the simultaneous hydrolysis of a nucleotide triphosphate One ATP is needed for each tRNA charging reaction

bacteria, eukaryotes

ribosome size: ______ 30S and 50S (70S) , ______ 40S and 60S (80S)

ribosome binding site

short sequence upstream of the start codon. Also referred to as a Shine-Dalgarno sequence located 3-9bp on the 5' side of the start codon (seq: 5'-AGGAGG-3'), is complementary to a sequence located near the 3' end of one of the ribosomal RNA (16s rRNA)

uORF

sometimes the first ORF is in a poor sequence context, resulting in its frequent bypass sometimes they're translated, and this allows interaction between initiation factors that tether the 40S subunit to the mRNA to be retained after termination. also scan next AUG after binding to a new initiator tRNA complex

uncharged

tRNA without amino acid is said to be ______

acceptor stem

the 3' end of a tRNA molecule; the portion that amino acids become attached to during the tRNA charging reaction pairs 5' and 3' ends of tRNA molecule (CCA) has discriminator base that can determine specificity of tRNA synthetase to another

P site

the binding site for peptidyl-tRNA

E site

the binding site for the tRNA that is released after the growing peptide chain has been transferred to the aminoacyl-tRNA

start codon

the first codon of a messenger RNA (mRNA) transcript translated by a ribosome 5'-AUG-3' but 5'-GUG-3' and 5'-UUG-3' can be used.

translation

the process by which ribosomes read the genetic message in mRNA and produce a protein product according to the message. One of the most energy costly processes in cell. Need mRNA's, tRNA's, aminoacyl-tRNA synthetases, and ribosome.

scanning

to find the start codon; go downstream from 5' end of mRNA ATP-dependent and stimulated by the eIF4A/B associated RNA helicase

polypeptide exit tunnel

where growing polypeptide is guided to surface of ribosome and ultimately released in 50S (large) subunit

AMP, 3'

2 steps of charging 1. Adenylylation in which AA reacts with ATP to become adenylylated with release of pyrophosphate. Transfers _____ to AA. 2. tRNA charging where AA-AMP reacts with tRNA, transfers AA to ___ end of tRNA.

proton shuttle

2'-OH of P site tRNA act as ________, 2'OH donates a hydrogen to 3'OH of peptidyl-tRNA and accepts a proton from attacking alpha amino group of A site-tRNA. (for peptide bond formation)

L-shaped

3-D structure of tRNA is an _______ tertiary molecule

RNA, proteins

50S/30S structure: _____ component in interior, _____ in periphery of ribosome

codon-anticodon

Correct __________ base pairing facilitates EF-Tu bound to the aminoacyl-tRNA to interact with the factor-binding center inducing GTP hydrolysis and EF-Tu release

eukaryotic initiation

Both use start codon and initiator tRNA and both form complex with small subunit that assembles on mRNA before addition of large subunit. Small subunit associated with initiator tRNA when recruited to 5' capped end of mRNA. Scans along mRNA in 5'->3' direction unitl it reaches the first 5'-AUG-3' (kozak sequence i think). Binding of initiator tRNA to small subunit always precedes association with mRNA Separate set of auxiliary factors mediates recognition of mRNA Small ribosomal subunit bound to initiator tRNA scans mRNa for first AUG sequence. Large subunit of ribosome recruited after initiator tRNA base pairs with start codon eIF1, eIF1A, eIF3, eIF5 bind to small subunit. Act in analagous manner to prokaryotic initiation factors and prevent large subunit from binding. Initiator tRNA escorted to small subunit by three subunit GTP binding protein eIF2. Only binds to tRNA in GTP bound state. eIF2 positions charged methionine (not N-formyl methionine) in P site, results in 43S preinitiation complex. Recognition of 5' cap by cap binding protein eIF4E. eIF4G binds both eIF4E and mRNA. eIF4B activates RNA helicase activity of eIF4A. Unwinds secondary structures. Finally recruit 43S preinitiation complex to mRNA to form 48S preinitiation complex

prokaryotic translation initiaton

Small subunit association mediated with base pair interactions on RBS and 16S rRNA. Need initiator tRNA, base pairs with start codon, has valine or methionine but Met-tRNA transformylase attaches a formyl group onto it, making it N-formyl methionine. Deformylase removes formyl group after synthesis is completed. IF-1 prevents tRNA from binding to portion of small subunit that will become A site IF-2 GTPase that interacts with components of initiation machinery. Interacting with subunits, IF2 facilitates association of fMet-tRNAfMet with the small subunit and prevents other tRNAs from associating with small subunit If3-binds to small subunit and blocks it from reassociating with a large subunit. Initiation requires a free subunit so the binding of IF3 critical for new cycle of translation.-occupies E site Last step is association of large subunit to small subunit (primed for initiation) forms 70S initiation complex.

initiator tRNA

Special tRNA that initiates the translation of an mRNA in a ribosome. It always carries the amino acid methionine. base pairs with start codon, either AUG or GUG coupled to N-formyl methionine to charge it (deformylase removes it)

domains, stabilize

The core function of ______ of the ribosome are composed either *entirely or mostly from RNA*. portions of some ribosomal proteins do reach into the core of the subunits, where their function seems to be to _______ the tightly packed rRNA by shielding the negative charge of the sugar-phosphate backbones Anticodon loops of charged tRNA's and codons of mRNA contact 16S rRNA, not ribosomal protein

complementarity

The extent of ___________ and spacing between the RBS and the start codon has a strong effect on how actively a particular ORF is translated. when high, and with proper spacing, translation favored

ribosome cycle

The large and small subunits undergo association and dissociation during each cycle of translation 1. Binding of the mRNA and initiator to the small ribosomal subunit 2. Recruits a large subunit 3. Protein synthesis (initiation) 4. Ribosome translocation from one codon to the next. (translation elongation) 5. Complete polypeptide release and ribosome dissociation

70S initiation complex

The last step of initiation involves the association with the large subunit to create a __________. In this step, IF3 is released, and the large subunit is free to bind to the small subunit. This interaction stimulates the GTPase activity of IF2•GTP. IF2•GDP has reduced affinity for the ribosome and the initiator tRNA, leading to the release of IF2•GDP as well as the IF1 from the ribosome

2 tRNAs

The ribosome has 3 binding sites for tRNA: to perform the peptidyl transferase reaction, the ribosome must be able to bind at least _____ simultaneously

initiation factors

With the binding of three ______, the small subunit is preparing to bind to the mRNA and the initiator tRNA. next step is association with large subunit to create 70S initiation complex

large subunit

_____ of ribosome: the peptidyl transferase center, which is responsible for the formation of peptide bonds

difference

______ between prokaryotic initiation and eukaryotic initiation: The small subunit is already associated with an initiator tRNA when it is recruited to the capped 5' end of the mRNA. The small subunit scans along the mRNA in a 5' to 3' direction until it reaches the first 5'-AUG-3'. (explains why the vast majority of eukaryotic RNAs encode a single polypeptide. More auxiliary factors are needed

mRNA recognition

______ by small subunit in eukaryotes: -recognition by 5' cap binding protein eIF4E -eIFG binds to both eIF4E and the mRNA -eIF4A binds eIFG and mRNA -eIF4B recruitment, which activate the RNA helicase of eIFA (to unwind the secondary structures that may have formed at the end of the mRNA)

small subunit

______ of ribosome: decoding center, in which charged tRNAs read or decode the codon units of the mRNA

cloverleaf

______ structure of tRNA because it has single-stranded and double-stranded regions, and many different types of loops from main stem acceptor stem, ψU loop, D loop, anticodon loop, variable loop

similarity

_______ between prokaryotic initiation and eukaryotic initiation: Both use a start codon, and a dedicated initiator tRNA, both use initiator factors to form complex with the small ribosomal subunit that assembles on the mRNA before addition of the large subunit