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 [iG1 6.04] [iG1 6.14b]
               Large Subunit (LSU) = 60S = 28S rRNA + 5.8S + 5S rRNA + 50 proteins
               Small Subunit (SSU) = 40S = 18S rRNA + 33 proteins
                                                       = 80S monosome

       A site (Aminoacyl), P site (Peptidyl), & E site (Exit) [APE complex]

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 2o modified 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) joined to COOH of amino acid(x)
       ~20 synthetase types 'recognize' correct anticodon loop
       isoacceptance:
           one-to-one correspondence between synthetase & amino acid


RNA Translation: Protein Synthesis


    Ribosomes "read" mRNA &  assemble polypeptide according to Genetic Code
              (online animation)

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

           tRNAfmet 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'

Elongation: addition of amino acids 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 tRNAphe enters A site

        peptide bond formed between fmet & phe
           P site amino acid transferred to A site amino acid      
                  uncharged tRNA released from P site (passes to E site)
                   amino end of  fmet remains unchanged ]

               and so on ...  [online MGA animation]
               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 with either purine / pyrimidine
                                Fewer tRNA species needed:
                       Ex.: three tRNAser 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'

Termination: release of polypeptide

       mRNA + tRNA(aan-...-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 tRNAn
                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 research, 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


Homework:


Practice DNA "Translation" problems [PDF download version]


All text material 2013 by Steven M. Carr