Introduction to Molecular and Cell Biology, Biol. 220

Lecture 7: The three roles of rNA.
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The Structure of RNA

Genetic information is stored in DNA.

Information transfer carried out by RNA.


Why use two different types of nucleic acids to store and transmit genetic program?

 

RNA Structure

What is the difference between DNA and RNA?

 

The nitrogenous base,Uracil, is used in place of thymine (Fig. 1) but it acts just like thymine in pairing with adenine .

Fig. 1Comparison of Thymidine and Uracil

The use of Ribose in RNA instead of Deoxyribose (Fig. 2).

Fig. 2Comparison of Ribose and Deoxyribose sugars

 

 

 In the chemical structure of ribose, at the 2' C there is a hydroxyl group instead of a hydrogen.

This leaves RNA less chemically stable with a tendency for hydrolytic cleavage, especially in strong base (Fig. 3). In contrast DNA is more stable.

Fig. 3Hydrolysis of the Phosphodiester bonds of RNA in the Presence of Strong Base


 

other Differences between DNA and RNA:

RNA is much shorter than DNA (recall that 1 DNA molecule spans the length of the entire chromosome. 

RNA may span a few thousand bases of DNA.  With a few exceptions ( a few viruses) RNA occurs in single stranded form.

 

The double-stranded nature of RNA

The nitrogenous bases can form intra-strand hydrogen bonds with complementary base pairs (Fig. 4). This is important in forming the secondary structure of RNA where stem/loop or hairpin structures form because the chains fold back on themselves, creating loops and small base-paired stretches between complementary regions.

Fig. 4 Intrastrand hydrogen bonding in RNA

 

One of the most remarkable and unexpected discoveries of recent years is that some folded RNAs have nucleolytic activity. The RNA enzymes, called ribozymes are able to cleave specific phosphodiester bonds in a manner analogous to protein enzymes.

 

Transcription

There are three classes of transcripts, messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA, (rRNA).

 

Primary Transcript

Fig. 5 Transcription and Translation

To view the transcription/translation animation you need a browser that supports the shockwave plugins.  

A primary transcript is copied from a sequence of DNA that codes for a specific protein (Fig. 5).

Messenger RNA (mRNA)

An mRNA is an RNA that is translated into protein.

 

Prokaryotes

In prokaryotic cells a primary transcript is used directly as an mRNA (often times before it is even completely transcribed).

In prokaryotes they are only around for a few minutes. Continuous synthesis of protein requires a continuous synthesis of mRNA. This helps the prokaryotic cell respond quickly to a fluctuating envirnoment and fluctuating needs.

The mRNA of prokaryotic cells is polycistronic (one  transcript can code for several different proteins) (Fig. 8).

Fig. 8. Polycistronic mRNA Transcription and Translation

Eukaryotes

In eukaryotic cells a primary transcript is processed before being exported from the nucleus as an mRNA:

  • A 5'CAP of 7-methyl guanosine is added (Fig.6).
    Fig. 6The 5' Cap Structure of Eukaryotic mRNA
  • A poly(A) tail is added to the 3' end of the transcript.
  • Introns (intervening sequences) must be cut from the transcript by a process known as RNA splicing (Fig. 7).
  • In eukaryotic cells the mRNA are stable for 4-24 hrs.
    •  

    The mRNA of eukaryotic cells is monocistronic (each transcript only encodes a single protein) (Fig. 5).

     

     

    The Genetic Code of mRNA

    The universal Genetic Code was deciphered in the early 1960s.
    It is a triplet code. In other words, each sequence of 3 mRNA nucleotides is a codon which corresponds to one amino acid.

    Example:

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

      N- Met    -  Phe  -  Arg  -  Thr   -C

     Each codon - codes for a specific amino acid or serves as a stop codon. (Stop codons are: UAA, UAG, UGA
    These three codons do not code for an amino acid. They signal as a stop signal for translation. )

     The Start codon is AUG.
    AUG codes for the amino acid, methionine, so every protein initially begins its sequence with methionine regardless of whether it is a prokaryotic or eukaryotic protein.

     There are 3 possible reading frames on a message depending upon where you start. So punctuation is very important (AUG) codon critical (Fig. 9).

    Fig. 9 Three possible reading frames (1-3) for each transcript and potential proteins. (Blue codons are for termination, red letters indicate potential polypeptides.

    1  ACG GGC ACA UGC AUG GCG AUU AAC UGA
                        M   A   I   N
    2 A CGG GCA CAU GCA UGG CGA UUA ACU GA
    3 AC GGG CAC AUG CAU GGC GAU UAA CUG A
                  M   H   G   D

     

     Because there are 4 different nucleotides, and it is a triplet code--  there are --  43=64 possible codon triplets for only 20 different amino acids. Therefore, most amino acids are specified by more than 1 codon-- in other words the genetic code is a degenerate code.

     An example, all of the following codons code for the amino acid arginine:

    CGU
      CGC
     CGA
     CGG
     The Genetic code also is highly conserved, the same in organisms as diverse as bacteria, plants, and man.

     There are as many different mRNAs as there are genes. Most proteins have between 100 and 1,000 amino acids so an mRNA must be at least between 300 and 3,000 nucleotide bases long.

    Each mRNA possesses codons that are read by tRNAs.

     

    transfer RNA

    -Small, ~ 70-90 ribonucleotides long (remember, they are transcribed from certain DNA sequences)
    -Single-stranded but folded into a particular 3D shape (Fig. 10).

    Fig. 10 Tertiary Structure of tRNA

    The 3D shape is held together by intrachain hydrogen bonds between complementary base pairs (Fig. 11).

    Fig. 11 Secondary Structure of tRNA

    Like all RNA molecules, they have a 3'-OH terminus, but the opposite end terminates with a 5' monophosphate rather than a 5'-triphosphate, because tRNA molecules are cut from a large primary transcript.

     see Figure of tRNA in text book (P. 274-275)

    Note the D arm which contains dihydrouracil, the T psi C arm which contains a thymine, a pseudouridine, and a cytosine. Also note the presence of the extra arm. In class I tRNAs the extra arm is quite small, in class II RNAs the extra arm is long.

     Note the 4 regions of molecule where you have intrastrand hydrogen bonding.

     Note the location of the anticodon. The anticodon is a sequence of 3 bases that can form base pairs with a codon sequence in the mRNA (Fig. 12).

    Fig. 12 tRNA Binding to mRNA, terminology

     No tRNA has anticodon complementary to stop codons UAG, UAA, or UGA

     tRNAs are initially made as part of a somewhat longer RNA transcript. First cut out, then completed by an enzyme that adds the three nucleotides CCA to the 3'end. Note that the 3' ribonucleotide base is ALWAYS adenine.
     

    Attaching Amino Acids to tRNAs

     The amino acid attachment site is located at the 3'OH terminus
    The amino acid, corresponding to the specific tRNA anticodon, is covalently linked to this terminus. Amino acids will come off to form polypeptide chain during protein synthesis.

     tRNAs are actually Amino acid carriers

     With the help of a specific enzyme the tRNA recognizes and binds to a specific amino acid.
    The enzymes are termed: aminoacyl tRNA synthetases (Fig. 13).

    Fig. 13 Synthetase Charging of tRNA with Amino Acid

     
    There is a different synthetase enzyme for every amino acid. A specific aminoacyl tRNA synthetase matches the amino acid with the anticodon; to do so, the enzyme must be able to distinguish one tRNA molecule from another. The necessary distinction is provided by an as yet ill-defined region encompassing many parts of the tRNA molecule called the recognition site. At the opposite end of the molecule is a loop which contains the anticodon. As the mRNA codons are read, only the appropriate tRNA can hydrogen bond with the codon by virtue of the anticodon. [See Figure 7.2 in text of how enzyme adds amino acid to the tRNA.]

    G A A  mRNA codon
       |   |   |
      C U U tRNA anticodons

     

     transfer RNA Terminology

    charged - tRNA with amino acid attached
    uncharged - tRNA, no amio acid
    mischarged - incorrect amino acid

     The different tRNA molecules and synthetases are designated by stating the name of the amino acid that can be linked to a particular tRNA molecule by a specific synthetase:
    i.e. leucyl-tRNA synthetase attaches leucine to tRNA^Leu

     Only the tRNA molecule, not its attached amino acid determines where the amino acid is added during protein synthesis. If you mischarge a tRNA it will put wrong amino acid in according to the anticodon.

    tRNA Tutorial for Phe-tRNA

     

    Ribosomal RNA

    The ribosome is the site where mRNA is translated into protein. It brings together the tRNAs, mRNAs, and specific amino acids.

    Classification and Component Structure

     Ribosomes are composed of 2 subunits a large subunit and a small subunit.

     Prokaryotic ribosomes differ from eukaryotic ribosomes as shown in Fig. 14. The ribosomes and ribosomal subunits are described in terms of their approximate rate of sedimentation (in Svedberg units). The higher the mass of the particle the higher the s value and the greater the rate of sedimentation.

    Fig. 14

    Prokaryotes
    Sedimentation (S) RNA (S) # Proteins
    30S 16S 21
    50S 23S
    5S    		 34
    combined = 70S
    Eukaryotes
    Sedimentation (S) RNA (S) # Proteins
    40S 18S 30
    60S 28S
    5.8S
    5S    		 45
    combined = 80S
    * S = Svedberg units which indicate the rate of sedimentation when centrifuged. Generally, the larger the number, the larger the particle.

     
    Function of Ribosomes

    The ribosome serves as the site of protein synthesis. mRNAs, tRNAs, and amino acids are brought together.

    On the ribosome, the mRNA fits between the two subunits (the interactions are stabilized by interchain hydrogen bonding). The tRNAs occupy a site on the large ribosomal subunit.

     The ribosome attaches to the mRNA at or near the 5'end.

  • In prokaryotes there is a ribosome binding site near the 5'end of the mRNA.
  • In eukaryotes, the ribosome first attaches at the 5'CAP (7-methyl guanosine).

    •  The ribosome then moves along the mRNA in the 5' to 3' direction, one codon at a time.

     

     
    Created 2004 by CA Rinehart• email CA Rinehart Index • CourseInfo LogIn • Syllabus • References • Other Resources