Translation- Exam II

Micro 4130 Exam II

Results of transcription

Mostly mRNAs that get translated into proteins, but also some functional RNAs like rRNA, tRNA, and tmRNA as well

Purpose of ribosome in translation

Decoding of mRNA and catalyzing peptide bonds between amino acids specified by mRNA

What ribosomes use

mRNA as template, amino acids linked to tRNAs as substrates, and protein as product

How amino acids are connected into proteins

Via a peptide bond between the carboxyl group of the first amino acid and the amino group of the second amino acid

N terminus

The free amino group on the beginning of the amino acid chain

C terminus

The free carboxyl group on the end of the amino acid chain

Primary structure of proteins

Amino acids linked in a single chain

Secondary structure of amino acids

Local interactions of a single protein chain creating a-helices or B-sheets

Tertiary structure of proteins

Interactions and folding of a single polypeptide chain, such as a-helices and B-sheets interacting

Quartenary structure of proteins

Interactions between multiple different polypeptide chains

Where does protein folding start

In the peptide exit tunnel of the ribosome

Components of translation

mRNA, Aminoacyl-tRNAs (aa-tRNAs), rRNAs, ribosomal proteins, initiation, elongation, and release factors, and recycling factors (RRFs)W

Why do all tRNAs hve almost the same 3-D structure?

Because they all have to fit in and interact with the inside of the ribosome

General lifecycle of a tRNA

Transcription, Maturation, Translation

Transcription of a tRNA

Creates a pre-tRNA that is much longer than the final product

Maturation of tRNA

End processing by adding a CCA, modification, folding, and some get spliced

Translation with tRNA

Aminoacylation, binding to EF-Tu and GTP to create the ternary complex, ribosome binding and mRNA decoding, and tRNA release

How amino acids are connected to tRNAs

Via an ester bond between the CCA at the 3’ end of the tRNA and the carboxyl group of the amino acid. The 3’ end of the tRNA is always longer

aa-tRNA

Aminoacyl-tRNA, an amino acid connected to a tRNA by an ester bond

Components of a tRNA

Acceptor arm/stem, T loop/arm, D loop/arm, Anticodon arm/stem, anticodon loop

T and D loops

Sites of the tRNA that get heavily modified, allowing the tRNA to be more stable and have 3-D structure

Where does the anticodon on the tRNA bind to the codon of the mRNA

In the small subunit of the ribosome

Intramolecular base pairing of tRNAs

Happens in the acceptor arm, d and t loops, and the anticodon arm to give the tRNA its structure

Stop codons

UAA, UAG, or UGA- the only 3 codons that don’t have a tRNA to bind to them and don’t encode for an amino acid.q

aa-tRNA synthetase

Use ATP to charge tRNAs with amino acids. Each amino acid uses a specific aa-tRNA synthetase. The amino acid is attached to the CCA at the 3’ end of the tRNA

Adenylation

The amino acid is activated with ATP in the synthetase active site, forming aminoacyl-AMP and releasing PPi.

Steps of the aminoacylation reaction

Adenylation, then transfer. This reaction is catalyzed by aminoacyl-tRNA synthetases

Transfer

The amino acid is transferred to the 3’-OH of the tRNA and AMP is released

Isoacceptors

tRNAs with different anticodons carrying the same amino acid

Aminoacylation

The process of charging an amino acid onto a tRNA. Is done by aminoacyl transferases

aa-tRNA synthetase specificity

These enzymes have extremely high specificity in their amino acid recognition sites because many of the amino acids look similar

Aminoacylation of tRNA process

Amino acid and ATP bind to the enzyme. Enzyme catalyzes adenylation of the amino acid, coupling amino acid and AMP, losing 2 phosphates. An uncharged tRNA binds to the enzyme, and the enzyme transfers the amino acid from aa-AMP to tRNA, forming aa-tRNA. Then both he aa-tRNA and AMP are released from the enzyme, and the enzyme returns to its original state

Requirements for accurate decoding

proper mRNA-tRNA codon-anticodon base pairing and proper tRNA-aa charging

aa-tRNA synthetase editing

Mistakes can occur in the process of aminoacylation, and these aa-tRNA synthetases can fix them in either pre- or post-transfer editing (before or after the aminoacyl group is transferred onto the tRNA).

Error rates in processes of the central dogma

Increase as you move from replication to transcription to translation. Fewer mistakes early on because they would have worse effects in the cell- errors in DNA replication can have catastrophic effects compared to mRNA transcripts and protein products that get made in many copies, so more flexible with mistakes

Codon chart

Lists the 3 bp mRNA sequence that complementary anticodons of tRNAs bind to.

Degeneracy of the codon code

Genetic code is redundant. There are 64 combinations of codons, but only 20 standard amino acids. 3 of the codons encode for stop instead of an amino acid

Wobble pairing

How the genetic code can have less than 61 tRNAs even though there are 61 codons that encode for an amino acid. Standard based pairs may be strictly used in the first two positions of the triplet, but the pairing of the third nucleotide may have more relaxed requirements.

Types of Wobble base-pairing

G pairing with U, or I pairing to U, A, or C.

Inosine

Found in tRNAs, adenosine gets deaminated to form this

Composition of the bacterial ribosome

rRNAs make up 2/3, and ribosomal proteins make up the last 1/3

50S subunit of the ribosome

Large subunit. The active site. Is the PTC (peptidyltransferase center), catalyzes peptide-bond formation

30S subunit of the ribosome

Small subunit. Binds mRNA and mediates selection of cognate aminoacyl tRNAs by facilitating base-pairing between mRNA codons and tRNA anticodons. Decoding!

70S bacterial ribosome

Composed of 50S and 30S subunits

Peptidyl tRNA

tRNA with the growing peptide linked to the 3’ nucleotide

Direction of ribosome

Reads mRNA 5’-3’, makes protein N- to C- terminus

E site

Site on the ribosome that binds the deacylated tRNA before it leaves the ribosome

P site

Site on the ribosome that binds peptidyl-tRNA

A site

Site on the ribosome that binds aminoacyl tRNAs

tRNA incoming to ribosome content

aa at 3’ end of tRNA, EF-Tu bound to GTP

How does the ribosome know where to start/end translation

Ribosome binds to RBS or Shine Dalgarno sequence. Translation begins at the start codon to set the reading frame and ends at a stop codon.

Shine Dalgarno

Sequence on mRNA that the ribosome binds to before starting translation. This is upstream of the start codon so it doesn’t get translated

3 models for translational coupling in operons

RNA unfolding model, 30S scanning model, and 70S scanning model.

IF3

Binds to 30S subunit, blocking E site. This releases previously translated mRNA and deacylated tRNA from the 30S. Also blocks re-association of 30S and 50S. Helps in selection of proper start codon (if it is incorrect it destabilizes the fMet-tRNA-30S complex.

IF1

Binds to block the A site.

Significance of IF1 and IF3 binding

Only the site that should be used by the tRNA (the P-site) is free.

rRNA binding to Shine Dalgarno/RBS

3’ of 16S rRNA binds to the SD/RBS, positioning the start codon in the P site for the binding of charged fMet-tRNA

IF2

Specific for charged fMet-tRNA. IF2-GTP stimulates association of the 50S subunit, then gets hydrolyzed to IF2-GDP, making the ribosome ready for elongation

Charged fMet-tRNA

The initiator tRNA- charged with formylmethionine (prevents association with EF-Tu and promotes recognition by EF-Tu

Formyl group on fMet-tRNAq

Formyl group added when Met is on tRNA. Formyl group prevents use in elongation, and only allows for use in initiation

Specificity of Shine Dalgarno

Ribosomes all almost have the exact same SD sequence. Variations in this determine how often the message is expressed.

Purpose of Shine Dalgarno

Located within 5-10 nucleotides upstream of the start codon, helps the correct positioning of the p site of the ribosome

Frequency of start codon variants

AUG is the most prevalent start codon, but there are others that the cell can use. GUG and UUG are the next most popular, but they are less efficient and don’t use watson crick base pairing

Regulation via start codons

GUG and UUG are less efficient at translation, and this can be used as a method of regulating translation

Structural differences in fMet-tRNA and Met-tRNA

Structural difference in the acceptor stem and anticodon stem, but the same anticodon sequence. IF2 only recognizes fMet-tRNA, and EF-TU only recognizes Met-tRNA

Results of IF1 and IF3 being released

Stimulates GTPase activity

Getting ribosome ready for the elongation cycle

IF1 and IF3 dissociate, IF2-GTP gets hydrolyzed. Charged tRNA can now bind in the elongation phase.

Overview of Intiation

30S subunit binds to the mRNA at the shine dalgarno site. IF1 and IF3 bind to the A and E site respectively so that only the P site is open for the initiator tRNA to bind to. IF2-GTP recognizes and binds to the initiator tRNA, and the IF2-GTP causes the 50S subunit to associate. IF3 and IF1 leave, and IF2-GTP gets hydrolyzed before leaving

3 major steps of elongation

Decoding, peptidyl transfer, and translocation

Decoding

aa-tRNA is delivered to the A site as part of a ternary complex with EF-TU and GTP. 30S subunit physically does this- is bound to the mRNA, and tRNAs bind here.

Ternary complex

Composed of EF-Tu, GTP, and aa-tRNA.

Peptidyl transfer

Immediately follows decoding, and is rapid and irreversible. Transfer of the peptidyl group from P-tRNA to A-tRNA.

Translocation

Catalyzed by EF-G-GTP. The coupled movement of tRNA and mRNA in the ribosome. Deacylated tRNA dissociates from the E site before or during the next round of elongation.

Process of elongation

tRNA with growing polypeptide chain is in the P site. EF-Tu-GTP carries the next charged tRNA to the A site. Ribosome recognizes the shape of the bp and rejects incorrect tRNAs. This complex entering the ribosome changes its conformation so that its GTPase activity is activated. EF-Tu-GTP undergoes hydrolysis and leaves, aa-tRNA is now free to react. Transfer of the growing peptide from the P-tRNA onto the A-tRNA. EF-G-GTP enters and catalyzes translocation, getting hydrolyzed and entering the A site. Uncharged tRNA leaves the E site, and EF-G-GDP leaves the A site

Movement of things through sites of ribosomes

Charged tRNAs enter the A site where they have the peptide chain transferred onto it. EF-G-GTP moves into the A site, stimulating translocation. Translocation moves the empty tRNA into the E site and the peptide-bound tRNA into the P site.

Mechanism of Translocation

50S subunit moves first, moving 3’ ends of the tRNAs down one site. EF-G binds to the A site, pushing the 30S end to follow. GTP gets hydrolyzed, causing the factor to fall off, opening the A site for the next tRNA to join

Termination overview

A stop codon in the mRNA A site recruits either RF1 or RF2 to mediate the hydrolysis and and release of the peptide from the tRNA in the P site. This functions as a signal to recruit RF3-GDP, which induces the release of RF1’2. Exchange of GDP for GTP on RF3 and subsequent hydrolysis releases RF3. The ribosome is left with mRNA and a deacylated tRNA in the P site. This complex is disassembled by the binding of ribosomal release factor RRF and the EF-G elongation factor. GTP hydrolysis causes the dissociation of the 50S ribosomal subunit, and initiation factor 3 (IF3) is required to dissociate the deacylated tRNA from the P site.

Where does RF1/2 bind

In the A site when there is a stop codon present

What breaks in termination of translation

The ester bond between the tRNA and the peptide

What codons does RF1 recognize

UAA and UAG

What codons does RF2 recognize

UAA and UGA

Most commonly used stop codon

UAA

RRF

Ribosomal recycling factor, comes in wit EF-G and hydrolyzes GTP, causing dissociation of the 50S ribosomal subunit

Polysome

Multiple ribosomes engaged on the same mRNA, translating the message several times at once

Txn and tln coupling in bacteria

Because no nucleus! So both can take place at the same time in the cytoplasm.

Antibiotics

Antibiotics can inhibit any given step of translation, which kills cells.

IF1 function in eubacteria

Block A site and bind mRNA

IF2 function in eubacteria

Initiate tRNA binding

IF3 function in eubacteria

Subunit dissociation

EF-Tu function in eubacteria

tRNA binding

EF-G function in eubacteria

Translocation

RF1 function in eubacteria

Termination

RF2 function in eubacteria

Termination

RF3 function in eubacteria

Release RF1/2