Lecture 4: Translation Flashcards by Zoe Phillips

Synthesized as large precursor tRNAs with regions of complementary binding, then
processed by special _____ & ______ to produce extensive
secondary structures (a.k.a cloverleaf structures) resulting from base-paired regions

exonucleases and endonucleases

How well did you know this?

Not at all

Perfectly

there are lots of post-transcriptional modifications done to tRNAs that generate odd/unusual bases… what are they?

Dihydrouridine (D)
* Fully saturated pyrimidine ring (no
double bonds)

Pseudouridine (ψ)
* Ribose joined to C#5 instead of N#1

Ribothymidine (T)
* Methyl group is added to C#5 of uridine

Methylguanosine (mG)

Modified purine (Y)

How well did you know this?

Not at all

Perfectly

T/F: Genes encoding tRNAs scattered throughout the chromosome

true!!
* May be present in rRNA operon
* May form an operon consisting of 2-7 different tRNA genes
* May be alone (i.e. monocistronic)

How well did you know this?

Not at all

Perfectly

what is the function of tRNA molecules?

Function to carry amino acids to the translation machinery
* Each tRNA contains a specific 3-nucleotide sequence called the anticodon, a
group of three bases that recognizes a codon (a 3-base code for an amino acid)

How well did you know this?

Not at all

Perfectly

how many amino acids are there?

20, plus 2 unusual

How well did you know this?

Not at all

Perfectly

how is the mRNA transcript read by translation machinery?

read 5’ to 3’ synthesizing 5’ to 3’

How well did you know this?

Not at all

Perfectly

codons are…

sets of three nucleotides, each specifies an amino acid

How well did you know this?

Not at all

Perfectly

T/F: the genetic code is degenerate

true!! 64 codons, only 20 amino acids

Thus, a codon can determine the corresponding amino acid BUT the amino acid
cannot determine the specific codon sequence

How well did you know this?

Not at all

Perfectly

what is the start codon? what does it code for?

AUG- methionine

How well did you know this?

Not at all

Perfectly

what are the stop codons?

UAA, UAG, UGA

How well did you know this?

Not at all

Perfectly

T/F: only 1 tRNA molecule for each amino acid = so at least 20
different tRNAs plus special tRNAs for stop codons

true!!

How well did you know this?

Not at all

Perfectly

what is the wobble base hypothesis?

Flexible basepairing (mostly due to base modifications) between the 3rd position of
the codon and the 1st position of the anticodon, allows for some mistakes in the transcription

How well did you know this?

Not at all

Perfectly

T/F: Rules for wobble
hard to predict

true

Bases in tRNA are
sometimes
modified altering
basepairing
* E.g. inosine
(modified purine
base) can pair
with any residue

How well did you know this?

Not at all

Perfectly

what are the two most important components of the tRNA structure?

Acceptor domain (3’ –ACC) → amino acid is carried by the 3’OH end of the tRNA
Anti-codon loop (central loop) → contains 3 bases (anti-codon) which determines
placement of a specific amino acid into the growing polypeptide chain

How well did you know this?

Not at all

Perfectly

3’ end of tRNA molecule has unpaired CCA
* Generally not encoded in the
tRNA gene- meaning its a post-transcriptional modification

how is this CCA added to the tRNA 3’ end?

Added sequentially one at a time
by CCA-adding enzyme using CTP
and ATP as substrates
* Cognate amino acid covalently
attached to “A”

How well did you know this?

Not at all

Perfectly

Linkage of correct amino acid (as per anticodon) to tRNA is facilitated by an ______

aminoacyl-tRNA
synthetase

How well did you know this?

Not at all

Perfectly

to link the correct amino acid, what do we need?

Requires specific contacts (i.e. D loop, anticodon, parts of acceptor stem) be made between
regions of the tRNA and the synthetase

How well did you know this?

Not at all

Perfectly

The binding of amino acid to 3’ CCA end of tRNA is mediated by the
aminoacyl-tRNA synthetase in two steps: what are they?

Activation of the amino acid by reaction with ATP
Amino acid + ATP <-> aminoacyl-AMP + P-P (pyrophosphate PPi)
* Aminoacyl-AMP intermediate formed remains bound to the tRNA
synthetase until collision with the appropriate tRNA molecule
- links activated amino acid to synthetase
Activated amino acid is bonded to the CCA stem of its tRNA to form a charged
tRNA
Aminoacyl-AMP + tRNA <-> aminoacyl-tRNA + AMP
Aminoacyl-AMP is now bonded to CCA stem of its tRNA, and exits synthetase

How well did you know this?

Not at all

Perfectly

what provides the energy to incorporate an amino acid into the growing polypeptide chain?

energy rich phosphate bonds charge the tRNA (ATP-AMP)

How well did you know this?

Not at all

Perfectly

_____ Carries information about protein structure

mRNA

How well did you know this?

Not at all

Perfectly

T/F: ~90% of genes in E. coli code for mRNA (i.e. structural gene)

true!!! super high coding density

How well did you know this?

Not at all

Perfectly

what are the three main sections of an mRNA?

leader sequence
coding sequence
trailer sequence

How well did you know this?

Not at all

Perfectly

______ of mRNA
* 5’ end of mRNA transcript
* Contains a specific ribosome binding site (or the Shine-Dalgarno sequence)

Leader sequence

How well did you know this?

Not at all

Perfectly

_______ of mRNA
* Information is organized in codons (i.e. sets of 3 nucleotides)
* Each codon is complementary to anticodon of a tRNA
* Specifies amino acid
* Generally, begins with AUG start codon encoding chemically modified methionine
called N-formylmethionine
* Ends with stop (i.e. nonsense) codon

Coding sequence

How well did you know this?

Not at all

Perfectly

______ of mRNA * Transcribed to make the 3’ end of mRNA * Sequence of trailer determines stability of mRNA * e.g. degradation impeded by secondary structure, modulated by RNA binding proteins in response to environmental stress signals, etc.

Trailer sequence

But deletion or addition of nucleotide in DNA sequence, when transcribed, can put the coding sequence “out of frame”, this is called what?

frame-shift mutation

what 3 types of proteins can there be? that play major roles in cell function

1. catalytic proteins (enzymes) * Catalysts for chemical reactions that occur in the cell 2. Structural proteins * Integral parts of the major structures of the cell (membranes, walls, ribosomes, etc) 3. Regulatory proteins * Control cell processes by variety of mechanisms including binding to DNA and affecting transcription (i.e. gene expression)

proteins are polymers of...

amino acids

______: * Linkage between the –COOH group of one amino acid with the amino nitrogen of the second amino acid with the loss of one water molecule

Peptide bond

T/F: A protein consists of one or more polypeptides * Thus, translation of a mRNA transcript will result in a polypeptide sequence which may be the final protein product or may be part of a final protein product (i.e. subunit)

true!!

______ → linear sequence of amino acids in a polypeptide chain that ultimately determines the folding pattern of the polypeptide, which in turn determines its biological activity

Primary structure

_______ of polypeptides is Generated by hydrogen bonding between oxygen and nitrogen atoms of two peptide bonds to either form alpha helix or beta sheet * A polypeptide can contain regions (i.e. domains) of alpha-helix and regions of beta-sheet structures

secondary structure

_________: Higher-order structures generated by interactions between the R groups of the amino acids in a polypeptide * Three-dimensional form dependent on hydrophobic interactions, with lesser contributions from hydrogen bonds, ionic bonds and disulfide bonds * Many proteins consist of two or more polypeptides (i.e. subunits) and thus show quaternary structure stabilized by various interactions and also by disulfide bonds formed between cysteine residues in two different subunits

tertiary structure

______: Biological polymerization of amino acids into polypeptide chains

translation

what are the three stages of translation?

initiation, elongation, termination

70S Ribosome * Composed of two protein/rRNA subunits, what are they?

1. 50S subunit * 5S rRNA (~130 nucleotides) * 23S rRNA (~3000 nucleotides) * 31 proteins 2. 30S subunit * 16S rRNA (~1500 nucleotides) * 21 proteins

A. Initiation complex of translation requires: (5)

* mRNA transcript * 30S and 50S subunits of ribosome * Initiation factors (IF-1, IF-2 and IF-3) * Initiator tRNA with N-formylmethionine (fMet-tRNAfMet) * Guanosine triphosphate (GTP) for energy

what is the conserved sequence of the ribosomal binding site on mRNA?

UAAGGAGGU Located within 3-9 nts of start codon, conserved consensus sequence UAAGGAGGU complementary to short sequence within 16S rRNA → promotes hydrogen bonding of mRNA to 30S subunit

the ribosome binding site positions the ribosome where?

at the start codon AUG

once the ribosome binds at the RBS, what happens next? (we're positioned at AUG codon)

Initiator tRNA N-formylmethionine placed in P site * Formation of tRNA carrying N-formyl methionine * Enzyme transformylase modifies Met carried by tRNAfMet * Adds formyl group to amino group of methionine

which enzyme adds the formyl group to methionine?

transformylase

once formylmethionine is added, what happens next?

Then 50S subunit is added to finalize ribosome assembly

what three regions does the ribosome complex have?

A (acceptor) - Entry site for subsequent charged amino acid-tRNAs P (peptide) - fMet-tRNAfMet initiation codon & peptide bond formation E (exit) - Release of used tRNAs

once the mRNA binds to the 30S subunit, what happens?

IF-3 prevents 30S subunit from binding 50S subunit prematurely IF1 prevents inadvertent loading of N-formylmethionine in A site 3’ end of 16S rRNA complementary binds Shine-Dalgarno consensus sequence at 5’ end of mRNA aligning start codon with P site of 30S subunit IF-2 binds GTP, then binds fMet~tRNAfMet and helps guide it to “P” site

what does IF-3 do when mRNA binds to 30S

prevents early binding of 50S

what does IF-1 do when mRNA binds to 30S

blocks A site so that N-formylmethionie isn't added early

what does IF-2 do when mRNA binds to 30S

binds to GTP, then fMet-tRNA and helps guide it to P site

what happens when the 50S subunit attaches?

IF-1 and IF-3 are released IF-2 cleaves GTP finalizing the positioning of fMet~tRNAfMet in P site Binding of the 50S subunit completes the ribosome complex

how does the elongation in translation work?

1. Binding of aminoacyl-tRNA to the A site * anticodon base pairs with codon in the A site Two translation elongation factors (EF-Tu, EF-Ts) and 1 GTP required

how do the elongation factors help with the elongation of translation?

Elongation Factors 1. Charged tRNA is complexed with EF-Tu-GTP 2. Correct pairing will promote hydrolysis of GTP releasing EF- Tu-GDP 3. EF-Tu-GDP is recycled back to EF-Tu-GTP by EF-Ts-GTP

___________ catalyzes peptide bond formation between carboxyl end of growing polypeptide carried by the P site tRNA and the amino end of the amino acid carried by the tRNA in the A site * Event is referred to as transpeptidation

Peptidyltransferase

In the 50S subunit, the _______ has the catalytic activity

23S rRNA i.e. a ribozyme

_______ uses its ribozyme activity to function as a peptidyltransferase to form peptide bonds between – COOH of polypeptide chain and NH2 of incoming amino acid

23S rRNA subunit of the 50S subunit

_________: * Growing polypeptide attached to tRNA in the P site is transferred onto amino acid in the A site * Amino group of the amino acid reacts with carboxyl end of the polypeptide * Energy is supplied by breaking the high energy bond between polypeptide and tRNA in the P site

Transpeptidation

what is the result of transpeptidation?

Polypeptide has increased by one amino acid and is now attached to tRNA in the A site

After peptide bond formation, EF-G-GTP enters ribosome and moves (i.e. translocates) both polypeptide-containing tRNA and mRNA from _____ to ______

A site to the P site

Translocation requires hydrolysis of _____ bound to _____, after which EF-G is released from the ribosome

GTP bound to EF-G

The deacylated tRNA in the ____ later exits the ribosome in conjunction with the incoming charged tRNA in ____

E site A site

Each elongation cycle includes: (3)

* Binding of charged amino acid-tRNA, transpeptidation, translocation * Adds 1 amino acid to growing polypeptide * Ends when the ribosome encounters a stop signal

when the ribosome encounters nonsense codons (stop), what do release factors recognize?

Release Factors (RF) recognize specific stop codons; RF1 for UAA or UAG, and RF2 for UAA and UGA

Model is that peptidyltransferase tries to transfer polypeptide chain to release factor instead of amino acid thereby triggering events to....

release polypeptide

Translocation by ____ causes dissociation of ribosome from mRNA aided by ribosome release factor (RRF)

EF-G

Several ribosomes can translate a single mRNA molecule simultaneously, forming a complex called a _____

polysome

Polysome increase both the ____ and _____ of translation because each ribosome makes a complete polypeptide

speed and efficiency

A defective mRNA that lacks a stop codon cannot be properly translated * May be due to mutation, defective mRNA synthesis or partial degradation of mRNA * Traps ribosome because release factor cannot bind due to lack of stop codon * ______ process frees ribosome

Trans-translation

Production of a small RNA molecule called ______ which mimics both tRNA (carries an alanine) and mRNA (short sequence) containing a stop codon

tmRNA

how does the tmRNA rescue a stalled ribosome?

Collides with stalled ribosome * Binds alongside defective mRNA * Protein synthesis proceeds by first adding alanine, after which translation continues on short mRNA portion (~10 amino acids added) * Added amino acid sequence signals protease to degrade protein * Disassociated ribosomes are re-used

_____ aid in the folding of proteins

Chaperones

In E. coli, ____ and _____ are ATP-dependent enzymes that bind to newly formed polypeptides and prevent from folding too quickly

DnaK and DnaJ

Improperly folded proteins are transferred to multi- subunit complexes GroEL and GroES to complete folding using ____

ATP

______: Found in all 3 domains of life with prevalence in methanogens * conversion of serine (Ser) to Sec occurs through a single step catalyzed by selenocysteine synthase (SelA), after tRNASec is aminoacylated by seryl-tRNA synthetase (SerRS) * is encoded by the stop codon UGA followed by selenocysteine insertion sequence element (SECIS)

selenocysteine (one of the two unusual amino acids)

______: Prevalent in methanogens * Encoded by UAG stop codon * Similar mechanism of insertion

pyrrolysine (the other unususal amino acid)

Lecture 4: Translation Flashcards

(72 cards)