RNA Translation

RNA Translation: RNA makes Protein

In principle:
Translation of messenger RNA (mRNA) takes place on ribosomes,
which include ribosomal RNA (rRNA),
with the help of transfer RNA (tRNA)

Structure of rRNA & tRNA

ribosomal RNA (rRNA)
       rRNA + ribosomal protein ribosomes [iG1 6.09]
        Structure of rRNA: stems & loops [iG1 6.05]
             stems: double-stranded (dsRNA)
             loops: single-stranded (ssRNA)

        Structure of eukaryotic ribosomes [iGen3 06-13] [iG1 6.04] [iG1 6.14b]
           Large Subunit (LSU) = 60S = 28S rRNA + 5S rRNA + 50 proteins
         Small Subunit (SSU) = 40S = 18S rRNA + 33 proteins
                                                 = 80S monosome

A site (Aminoacyl), P site (Peptidyl), & E site (Exit) [iGen3 06-14]

transfer RNA (tRNA)
       the adaptor molecule: ~30 tRNA types
       2-dimensional 'cloverleaf' model [iG1 6.10]
            small: 75 ~ 90 nucs
       stems & loops
            D-loop & TC-loop ( = pseudo-uridylic acid)
                   tRNA characterized by 2^omodified bases [iG1 6.19]
            amino-acceptor stem
                    3' end is CACCA - 3'
                    5' end is G    - 5'
            anticodon loop
                   specificity of tRNA determined by 3-ribonucleotide sequence

3-dimensional structure is an "L" [iG1 6.12]
D- & TC-loops fold back on each other

Charged tRNA: aminoacyl synthetase_(x) forms ester linkage between
       3'-A of amino-acceptor stem of tRNA_(x) & COOH of amino acid_(x) [iGen3 06-10 , -11]
       ~20 synthetase types 'recognize' correct anticodon loop
       isoacceptance:
           one-to-one correspondence between synthetase & amino acid
           A "second genetic code"?

RNA Translation: Protein Synthesis

A three-step process (Review)
Ribosomes "read" mRNA & assemble polypeptide according to Genetic Code
(online MGA2 animation)

(1) Initiation at start codon (AUG) [iGen3 06-15]
              SSU binds at Shine-Delgarno sequence (-6 nucs) [iGen3 06-16]
              Initiation complex consists of mRNA, ribosome, & tRNA
         Multiple complexes form on a single mRNA: polysome (polyribosome)

tRNA_fmet always added first [N-formyl-methionine in prokaryotes]

In simplified form,

   5'-AUG-3' codon in mRNA
      |||
   3'-UAC-5' anticodon in tRNA

5'-CAU-3' if anticodon is written 5' 3'

(2) Elongation: addition of amino acids [iGen3 06-17, simplified] according to Genetic Code

Amino acids are joined via peptide bonds (see next section)
         Think of mRNA as fixed: ribosome moves along it 5'3'
                peptidyl (P) site on 5' end,
                aminoacyl (A) site on 3' end

        first AUG codon [for met] is in P site
            second UUC codon enters A site
                corresponding tRNA_phe enters A site

       peptidyl transferase forms peptide bond between fmet & phe
           P site amino acid transferred to A site amino acid [iGen3 06-18]
           tRNA_fmet ester bond broken, peptide bond to tRNA_phe formed
              uncharged tRNA released from P site (passes to E site)
               amino end of fmet remains unchanged

       and so on ... [online MGA animation] [iGen3 06-19]
               growing polypeptide in P site joins single amino acid in A site
               initial fmet always remains unchanged

   "Wobble": pairing of codon / anticodon goes 5'3' on codon
                      last position can miss-pair
                            Fewer tRNA species needed:
                       Ex.: three tRNA_ser species for six codons

tRNA Anti-codon	Alternative Serine mRNA codons
3'- AG G -5'	5'- UC C / U -3'
3'- AG U -5'	5'- UC A / G -3'
3'- UC G -5'	5'- AG C / U -3'

(3) Termination: release of polypeptide [iGen3 06-20]

^mRNA^{+ tRNA}(aa_n-...-aa₃-aa₂-aa₁)

^here^{: mRNA + tRNA}(lys-pro-gly-phe-fmet)

      stop codon (UAG, UAA, or UGA) enters A site
                no corresponding tRNA:
                release factor cleaves polypeptide from terminal tRNA_n
                polypeptide product is:   lys - pro - gly - phe - fmet

interactive translation animation [Genetic Science Learning Center, Univ Utah]
A talkie animation of transcription & protein synthesis

Griffiths et al. (1996) Fig. 13-7 is a nice summary (HOMEWORK)

Bioinformatics of DNA, mRNA, & Protein

5'- G T A A T C C T C - 3' DNA sense strand

5'- G U A A U C C U C - 3' mRNA

N - val - ile - leu - C protein

This is a logical, not a biochemical, relationship:
        Because mRNA is transcribed from the template strand,
        it "looks like" the sense strand (except for 'U').
        The information content of the DNA sense strand and mRNA are identical

Protein sequences can be read directly from DNA:
         Read the sense strand in the 5'3' direction,
         Substitute 'T' for 'U' in the code table [or in your head]
       Computer programs (Chromas, Sequencher, etc.) do this automatically

There are six possible ways of reading a piece of dsDNA
two 5'3' strands X three reading frames in each strand
Open Reading Frames suggest protein sequences

Deducing protein sequences from random DNA sequences is a major research activity
Bioinformatics: extraction of information from large macromolecular datasets

The following clues are useful:
         Remember that all prokaryotic coding sequences:
            are read only in the 5'3' direction
            begin with a "start" (AUG) codon
          end with a "stop" (UAG, UAA, or UGA) codon.
             Ex.: a typical exam problem is to identify a polypeptide of six amino acids from a dsDNA molecule

          But: in real life, your cloned eukaryotic DNA fragment
                may not have start or the stop codon for a complete protein,
                   [and not all AUG codons are 'start' codons]
                and may be partly or entirely an intron with one or more 'stop' sequences.

Extraction of information from random DNA sequences is a major activity of genomics

Do not assume that a dsDNA molecule is read from left to right, on the top strand

Suggested problems for review

MGA2, pp. 86-87
Solved problem 1
Problem ## 1, 2, 3

Practice DNA "Translation" problems [PDF download version]