CHAPTER 4, Part 2: FLOW OF GENETIC INFORMATION
Biochemistry 461

Notes(pdf)

Notes(Powerpoint)

• RNA properties in general
• mRNA properties
• Transcription:  RNA Polymerase
• Genetic Code
• Eucaryotic genes:  structural organization

• Genes (DNA) specify the amino acid or nucleotide sequences of functional cellular proteins and RNAs.



This can be represented schematically as:

                                                     transcription                           translation
Replication <=====> DNA  ==================> RNA   ---------> protein
                                       (<===transcription (reverse*)

Information Flow

*Recall that we know that RNA  ===> DNA flow of genetic information can occur (retrovirus reverse transcriptase)
 

• prions  - possible cause of CNS diseases(exs: kuru, scrapie, Kreutzfeld-Jacob disease) - So far, only protein particles without nucleic acid have been found.

 
• The genetic code consists of consecutive three letter(bases) words (codons) in DNA in mRNA which specify the sequence of amino acids in the primary structure of proteins.

 
• Codons in mRNA are "read" sequentially by anticodons in transfer RNA (tRNA) during protein synthesis. The mRNA is read in the 5' to 3' direction.

 
• Protein synthesis occurs on ribosomes (complex ribonucleoprotein particles).

 
• Most eucaryotic genes consist of both coding (exons) and non-coding (introns) information. The entire gene is transcribed, the introns are removed, and the exons are correctly joined together (spliced) during mRNA maturation.

 
• Evolutionary implications of introns and exons are extraordinary, since exons can be shuffled and rearranged to increase the diversity of protein structure and function.A) RNA

A)  RNA
 
• Four bases (A, G, C, U), ribose phosphate; nucleosides joined by 3' 5' phosphodiester bond (like DNA).

 
• Base-pairing is significant in most RNA molecules. RNA is usually single-stranded, but it can form base-paired hairpin loops (reversing 5' 3' orientation) with itself. A pairs with U and G pairs with C; base paired structures where the U of RNA pairs with A of DNA and G-C pairs also occur [Fig. 5-19]
[Fig. 5-28]. RNA can also form other complex structures.
 
• Cells contain 3 major types of RNA called mRNA (5%), tRNA (15%), and rRNA (80%) [Table 5-2].

 

• Infect E. coli cells with T2 phage and add ³²P-phosphate at same time.

Observe properties of polynucleotides which incorporate ³²P-phosphate

Studies of RNA synthesized in T2 bacteriophage-infected E.coli cells verified all predicted properties.

• Heavy isotope (¹³C, ¹⁵N) labeled E. coli cells were transferred to normal (¹²C, ¹⁴N) isotope media. Also ³²P (to label RNA) and ³⁵S (to label proteins) were added. Results were that RNA was synthesized after infection and translated on pre-existing (heavy) ribosomes. [Fig. 5-7]

 
• T2 mRNA formed base-paired double-strands (shown by DNA-RNA hybridization protocol) with T2 DNA, proving that it was copied from T2 DNA (Note: This hybridization method is important.) [Fig. 5-7, Fig 5-26]

Genetic Flow

Jacob and Monod predicted these properties:

        (1)   a polynucleotide [RNA]
        (2)   base composition complementary to a DNA template
        (3)   variation in size to reflect the variety of protein sizes [3 bases/amino acid]
        (4)   transient association with ribosomes
        (5)   rapid turnover  (about 2 minutes halflife in E. coli)

B) TRANSCRIPTION

• mRNA, rRNA and tRNA are complementary to genomic DNA (also shown by DNA-RNA hybridization experiments).

 
• All cellular RNA is synthesized (transcription of DNA) by DNA-dependent RNA polymerase enzymes which require:

 
(a) A template DNA, preferably double-stranded
(b) Activated precursors (all 4 rNTPs-ATP, GTP, CTP, UTP)
(c) Mg⁺⁺ in vivo

• RNA polymerase reaction is: [Fig. 5-25]
(RNA)_n + rNTP ===>   (RNA)_n+1 + PPi
• [as for DNA polymerase, the mechanism is a nucleophilic attack of free 3'-OH of primer on the -P of the rNTP substrate. PPi also drives the reaction forward here (PPi to 2Pi)]
• NOTE: RNA polymerase does not require a primer.
• RNA polymerase makes an RNA which is complementary to the DNA template strand.

 
• Transcription begins and ends at specific sites (relative to the template strand) called, respectively, promoters and terminators. Base sequences occur at these sites which are common to many different genes. These sequences differ between procaryotes and eucaryotes. [Fig. 5-32]

 
(a) Conserved promoter sites (2) are 5' to the start of transcription:

1) procaryotes: -10 (TATAAT) and -35 (TTGACA)
2) eucaryotes: -25 (TATA) and -75 (CAAT)
(b) Terminators have (in the mRNA) a GC-rich region followed by a string of U's which can form a base-paired hairpin loop. [Fig. 5-28]

• Transfer RNA (tRNA) is the "adaptor" molecule which brings amino acids to mRNA for translation of the codons into unique polypeptides.

 
• tRNA has two business ends [Fig. 5-30 & 5-31] :
(a) 3'-CCA_OH which accepts specific amino acids
(b) anticodon which base pairs with specific codon

• Genetic Code [Table 5-5, pp. 136]

 

b) The codon assignments were established using in vitro experiments with synthetic mRNAs. These mRNAs were made by enzymes or by organic chemical methods. [Fig. 5-16]

c) The genetic code is sequential and nonoverlapping.

d) The code is degenerate (more than one codon per amino acid); some codons are stop and start signals. [Table 5-4, Fig. 5-32 ]

e) The code is nearly universal [Table 5-4]. An exception, for instance, is use of UGA (a universal stop codon) as a code word for tryptophan in human mitochondria.

f)The sequences of genes and their encoded proteins are colinear - shown by Yanofsky experiments with polar mutants of -chain of tryptophan synthetase [Fig. 5-26].

D) STRUCTURAL ORGANIZATION OF EUCARYOTIC GENES

Procaryotic/Eukaryotic Genes

• Most eucaryotic genes (-globin, for instance) consist of coding sequences (exons) interrupted by non-coding (introns) sequences. [Fig. 5-33]
. Prokaryote genes are continuous.
 
• After transcription, the introns are removed and the exons are joined by splicing. Conserved sequences are found at splice junctions. The process is extremely accurate [Fig. 5-34]. Also following transcription, the mRNA is modified [Fig. 5-29].

 
• If there are 3 exons in a gene, they are (from 5' to 3') in the same order as they occur after removal of introns (i.e., polarity and order are maintained).

 
• Exons can code protein domains which are distinct functional units and which can be rearranged to give proteins with new functions. [Fig. 5-36]

 
• Different proteins may also be derived from a given mRNA by splicing in more than one way (ex: membrane-bound vs. soluble antibody). [Fig. 5-37]

 
• EVOLUTION: The RNA world - RNA came before DNA?

 
• Almost all eubacterial genes lack introns. They may have had them earlier in evolution and lost them while adapting to very rapid growth?

 
• RNA could be older than DNA and could have been able to replicate without proteins. Some RNA has self-splicing catalytic activity, hence it is a true enzyme (ribozyme). DNA could have finally evolved from reverse transcription of RNA. The primordial RNAs would have developed diverse "ribozyme" activities. [p. 115]

 
• Storage of genetic information as DNA is thermodynamically more favorable - DNA is more stable than RNA and fewer errors are made in its synthesis.

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Last updated by Don P. Bourque on September 9, 2002