0:02 Typically we think of ukareotic
0:04 translation elongation to be very
0:06 similar to the one that we have seen in
0:08 procariats. So you should definitely
0:10 watch that video before continuing this
0:13 one. The link to the complete
0:15 translation playlist is down in the
0:17 description as well as in the cards on
0:19 the top right corner. In proaryotic
0:22 translation elongation, we saw two main
0:27 elongation factors EFTU and EFG that
0:28 were involved in the moving of the
0:32 ribosome on the mRNA. In ukareiats,
0:34 there are two elongation factors that
0:37 are equivalent to the ones found in
0:40 procaryots. So EFTU has an orthologue
0:44 named ukareotic elongation factor 1A and
0:47 the EFG has an orthologue named
0:50 ukareotic elongation factor 2. In this
0:52 video we will discuss these two
0:55 elongation factors and how they work and
0:57 we will also see how hoostine base
0:59 pairing is involved in the translation
1:02 process. So we start where we left off
1:04 at the end of the initiation which is
1:08 the ads ribosome complex where the aug
1:10 start codon is bound by the initiator
1:14 tRNA that carries the methionine. So you
1:16 have the P site occupied by the transfer
1:19 RNA and the A site and the E site are
1:22 both empty. And as we saw in proariats,
1:25 the 60S subunit contains the factor
1:28 binding region. And this complex moves
1:31 on to the next step where the amino
1:34 acillated tRNA has to position itself at
1:37 the respective codon at the A site. And
1:39 this amino acillated tRNA is carried by
1:42 the elongation factor 1A which means
1:44 that the transfer RNA and the elongation
1:48 factor form a turnary complex. This
1:50 turnary complex therefore has the
1:53 elongation factor 1A which just like
1:56 EFTU carries a GTP and you also have the
1:58 transfer RNA in this complex. Let's
2:01 actually draw out this terinary complex.
2:05 The elongation factor 1A binds near the
2:07 threep prime end of the transfer RNA
2:09 close to the amino acid. Before we move
2:11 on, I just wanted to highlight that this
2:14 elongation factor 1A. The promoter of
2:17 this gene that encodes this factor is
2:20 quite frequently used in cloning where
2:22 some ectopic gene which has to be
2:24 expressed in mamalian cells is actually
2:27 driven by the promoter of EF1A.
2:30 And this factor EF1A is also sometimes
2:32 called elongation factor 1 alpha or
2:36 simply E F1 alpha. Now let's draw out
2:38 the complex that results when elongation
2:41 factor alpha with its transfer RNA binds
2:44 the codon at the A site. The elongation
2:47 factor 1A just like EFTU in procaryots
2:49 is positioned near the factor binding
2:52 region. And just for your note, the E
2:55 site is still not occupied by anything.
2:57 One detail worth mentioning at this step
3:00 is that the codon and anti-codon pairing
3:02 is partially controlled by the 18S
3:05 ribosomal RNA which is part of the 40S
3:08 ribosome subunit. Specifically the ATS
3:12 ribosomal RNA stabilizes the codon and
3:14 anti-codon pairing and it is the
3:16 secondary structure that is formed by
3:19 the ribosomal RNA and specifically at
3:21 the adnines in that secondary structure
3:24 that reach out into the major and minor
3:27 grooves of the anti-codon codon pairing
3:30 to help in the overall stabilization. So
3:32 if we simply draw this out, what we
3:35 notice is that when the pairing of mRNA
3:37 and tRNA is established, the 18S
3:40 ribosomal RNA is also trying to somehow
3:43 pair within this region at the A site.
3:45 It is not complimentary pairing, but it
3:48 is an interaction of adinine bases into
3:52 the codon and anti-codon pairs. And this
3:54 looks a lot like the triple helix
3:56 hydrogen bonding, which is based on the
3:58 principles of non-cononical base
4:00 pairing. one of which is known as the
4:03 Hoostein base pairing. These base
4:05 pairing mechanisms are special and
4:07 probably deserve to be addressed in a
4:09 separate video, but you should be
4:12 mindful of such subtleties in biological
4:15 processes. So when the anti-codon and
4:17 codon pairing is established and
4:20 stabilized, the SRL region in the factor
4:23 binding region activates the GTPAS and a
4:26 phosphate is released from the EF1A
4:28 factor. This causes the elongation
4:31 factor 1A to be in a complex with GDP
4:35 and as a result the EF1A is released. In
4:39 the case of EF1A, the conversion of GTP
4:41 to GDP actually causes a structural
4:44 rotation in the elongation factor 1A
4:47 complex. So it is the rotation which
4:50 releases the elongation factor 1A out of
4:52 this complex and not simply because it
4:54 just loses the affinity. And we know
4:56 that this is the rotation because some
4:59 specific translation inhibitors are
5:01 known to target the region which causes
5:03 the rotation of the elongation factor
5:07 1A. Dademian B is one such known
5:10 inhibitor. Anyways, the proper pairing
5:12 of the codon and anti-codon, the
5:14 accommodation mechanism that we talked
5:16 about in procaryots to position the tRNA
5:19 and the release of the elongation factor
5:22 1A brings the amino acid on a site tRNA
5:25 close to the amino acid on the P site.
5:28 And now we can draw out the next step.
5:29 And you notice that the amino acid on
5:32 the tRNA's at the pite and a site are
5:35 very close to each other. The e site is
5:38 still empty. All right. Now we also know
5:40 from the translation introduction video
5:43 that the 60S subunit contains a 28S
5:46 ribosomal RNA and like the 23S ribosomal
5:50 RNA and procariats the 28S ribosomal RNA
5:52 is responsible for peptide bond
5:55 formation in the ukareots. Now sometimes
5:57 under special condition the peptide bond
5:59 formation may require additional
6:02 translation elongation factors which in
6:05 ukarots is known as elongation factor 5a
6:09 which binds at the e site but largely
6:11 its main role appears to be in ensuring
6:14 efficiency and fidelity of the ribosome.
6:17 So mostly it has regulatory functions.
6:20 Oftentimes proline repeats need help
6:23 from elongation factor 5A but otherwise
6:26 it is not as essential in procariats.
6:28 Similar function is done by elongation
6:31 factor P. The overall process and the
6:34 mechanism of peptide bond formation is
6:37 similar in both procariots and ukareots
6:40 which we talked about in the proarotic
6:42 elongation process and this was
6:44 described by the proton shuttle and the
6:47 proton wire mechanism. If you're
6:48 interested in these mechanism, you
6:50 should definitely check out the
6:52 translation elongation step in
6:54 procariats for details. The link to that
6:57 video is down in the description.
6:59 Following the peptide bond formation, we
7:02 can draw out the resulting structure.
7:04 And what we notice, unsurprisingly, is
7:07 that the methunine from the pite tRNA is
7:10 transferred onto the tRNA at the A site.
7:12 The E site is still empty. Essentially
7:15 all we have done is formed a peptide
7:17 bond between the two amino acids. And
7:20 now this complex is ready for the next
7:22 step which is called transllocation. And
7:25 this step of transllocation needs the
7:27 second elongation factor elongation
7:30 factor 2 which just like EFG in
7:33 proariots comes with a GTP attached to
7:35 it. But there's a small difference in
7:38 ukareotic orthologue because EF2
7:40 contains a post translational
7:43 modification at one end of the protein
7:45 which is known as diptomide which is
7:47 just a chemical modification of the
7:50 histadine amino acid on the protein and
7:52 this dipomide modification is often
7:55 times required for breaking the 18S
7:57 ribosomal RNA interaction with the mRNA
8:00 and the tRNA at the A site. So in this
8:04 ATS ribosome the EF2 positions itself at
8:06 the factor binding region where the
8:09 daptoide modification is close to the
8:12 codon anti-codon pairing region and as
8:15 we saw in the procariats the aight tRNA
8:17 because it has the amino acid chains
8:19 wants to naturally move to the pite
8:21 because there's a protein exit channel
8:24 which is very close to the pite. So this
8:27 causes the tRNA to move and as a
8:31 feedback the 40S subunit rotates and
8:33 this causes the chain to move into the
8:35 protein exit channel. The energy for
8:37 this movement is provided by the
8:40 phosphate bond present in the GTP of the
8:42 elongation factor 2. So after
8:45 transllocation, the AUG start codon
8:47 which was at the P site now moves into
8:50 the E site and the A site codon moves
8:53 into the P site and as a result the
8:56 associated transfer RNA also move with
8:58 them because they're paired with the
9:01 codons and now the amino acid chain can
9:03 go into the protein exit channel. So the
9:07 ribosome has moved by one codon and the
9:11 AITE is occupied by the EF2 GDP complex.
9:14 This transllocated ribosome now moves to
9:16 its original position and the 4DS
9:19 subunit rotates back which causes the
9:22 EF2 GDP which anyways has low affinity
9:24 because of its loss of phosphate from
9:28 the GTP to be released and the eite tRNA
9:31 is also released. So in the next step
9:34 the P site is the only occupied site
9:36 where the amino acid chain is linked to
9:39 the transfer RNA and both E and A sites
9:42 are empty. This situation is exactly
9:45 like the starting ADS complex except
9:47 that the amino acid chain is a bit
9:50 longer and this is ready for the next
9:52 amino acillated transfer RNA to be
9:53 brought in by the elongation
9:56 [clears throat] factor 1A. And with each
9:58 cycle of translation elongation, the
10:01 amino acid chain will continue to grow
10:03 until the A site encounters a stop
10:06 codon. And when a stop codon is
10:08 encountered, the ribosome under goes the
10:11 termination step which will be the next
10:14 video. Now before you leave, here's a
10:16 cool interesting twist in ukarotic
10:19 translation. In fungi specifically, they
10:21 have an elongation factor known as
10:25 elongation factor 3 that binds the eite
10:28 tRNA and causes it to release. This is
10:31 special to only fungi. Us humans and
10:34 other higher order ukareots don't have
10:36 this factor. It's interesting how many
10:38 things in translation are conserved
10:41 across species but fungi have their own
10:44 little trick. And this wraps up our
10:46 discussion for translation elongation
