CHAPTER 3

RNA is the initial product of all genes. It differs from DNA in that it uses the 5 carbon sugar ribose in its nucleotides and doesn't use Thymine but instead uses Uracil (U). It is typically single stranded. Fig 3-3. RNA molecules may be informational in that their nucleotide sequence and translated into an amino acid sequence thereby forming a polypeptide. Such informational RNA molecules are referred to as messenger RNA (mRNA). Alternatively, the RNA molecules may be functional and categorized as:

Transfer RNA (tRNA)- molecules that transport amino acids to ribosomes where they are used during the translation of mRNA into a polypeptide.

Ribosomal RNA (rRNA)- form components of the ribosomes.

Small nuclear RNAs (snRNAs)- small RNA molecules which remain in the nucleus of eukaryotes and function in splicing RNA. They combine with small nuclear proteins and form snRNPs when involved in splicing.

Small cytoplasmic RNAs (scRNAs)- Combine with polypeptides that are destined for secretion, when they are being synthesized, so that they will enter the lumen of the endoplasmic reticulum.

TRANSCRIPTION

For any individual gene only one of the DNA strands serves as the template, the other is referred to as the nontemplate. Along the same DNA molecule one strand may be the template for one genetic locus and the other strand the template at a different locus Fig. 3-4. The template is always read in a 3' to 5' orientation by enzymes when synthesizing nucleic acids  Fig. 3-5. When discussing locations along the DNA we always refer to the order of the nontemplate strand.

The enzyme that transcribes the DNA into RNA is called RNA polymerase. There is only one RNA polymerase in prokaryotes but 3 in eukaryotes.

RNA polymerase I transcribes rRNA
RNA polymerase II transcribes mRNA
RNA polymerase III transcribes tRNA

RNA is always transcribed (synthesized) in a 5' to 3' direction. Fig.3-5.

Elongation- Ribonucleoside triphosphates are added to the growing RNA strand by cleaving their terminal diphosphates and attaching the nucleotides 5' end to the 3' end of the RNA strand. The energy for this is provided by the removal of the phosphates.

Termination- In prokaryotes the termination of transcription may be signaled by a protein (rho) that attaches to the growing RNA and follows the RNA polymerase in a 5'to3' direction. When the RNA polymerase stalls at a GC rich region of DNA the rho catches up with the polymerase leading to termination. Alternatively termination may be signaled in both prokaryotes and eukaryotes, by a sequence of approximately 40bp. This sequence has a stretch of GCs followed by 6 or more A's on the template. The GC region codes for RNA that forms a hairpin that is recognized by the RNA polymerase as a termination signal. The A's pair with complementary U's on the RNA weakly because they only have two hydrogen bonds per pair, therefore represent an easy region where the RNA and DNA may be separated Fig 3-10.

Following transcription the eukaryotic RNA is processed prior to leaving the nucleus. The processing of pre-mRNA is:

First a 7-methylguanosine cap is attached to the 5' end of the transcript via a triphosphate bond.

Second, a AAUAAA sequence on the RNA is recognized and the RNA is cleaved 20 nucleotides downstream.

Third, a 150-200 base long poly-A tail is attached to the 3' end.

Fourth introns are removed and exons spliced together. Fig.3-11.

Splicing is done somewhat differently for pre-tRNA, pre-rRNA and pre-mRNA. We will consider the pre-mRNA molecules. Virtually all introns possess a 5'GU sequence on the 5' end of the intron and a AG3' on the 3' end of the intron. Upstream between 20-50 bases from the 3' end of the intron is an A. snRNA molecules combine with proteins forming snRNPs (small ribonucleoprotein particles) which catalize the cutting and splicing reactions. First, the 5' end of the intron is cleaved from the upstream exon. The 5' end of the intron is then attached to the 2' carbon of the intermediate A in the intron which is called the branch site. Next the 3' end of the intron is cleaved from the 3' exon, thereby releasing the intron as a lariat shaped molecule. The 5' exon and the 3' exon are then ligated together. This involves a set of snRNPs which together form a structure referred to as a spliceosome Fig 3-12, 13, 14, 15..

Some RNAs are capable of catalyzing their own splicing. They are called ribozymes and are commonly found in pre-rRNAs. Chemically two nucleophylic reactions occur, the first between guanosine and the primary transcript (see Fig. 12-13*). The 3'-OH group of guanosine is transferred to the nucleotide adjacent to the 5' end of the intron. The second reaction involves the newly acquired OH group on the upstream exon and the 3' end of the intron. The intron is spliced out and the two exons are ligated, leading to a spliced gene.

PROTEIN STRUCTURE

Primary structure- sequence of amino acids

Secondary structure- repetitive structure due to hydrogen bonds forming between regions of the carbon-nitrogen backbone of the polypeptide. Forms alpha helix, etc.

Tertiary structure- Three dimensional structure formed by bonding between side chains.

Quaternary structure- Emergent structure resulting from more than one polypeptide binding together.

Remember that amino acids all have an amino and carboxyl group.

Proteins may be globular (e.g. enzymes or antibodies) or fibrous (structural).

The three dimensional shape of the polypeptide is important because it generates the active site (i.e., the region where the enzyme binds with the substrates).

TRANSLATION

The mature mRNA that leaves the nucleus of eukaryotes attracts the two ribosomal subunits and the nucleotide message is translated into the primary structure of the polypeptide. The tRNA molecules play a vital role in this translational process. The mRNA is read in a 5' to 3' direction 3 nucleotides (a codon) at a time. A sequence of three nucleotides in a loop of a tRNA molecule (the anticodon) will hydrogen bond with 3 complementary nucleotides of a codon. This takes place in a ribosome. The sixty four possible triplet codons contain messages for 20 different amino acids and stop signals (fig. 3-18). The tRNA is said to be charged, when an amino acid is bound to its 3' end. Amino acids are bound to tRNAs with the assistance of enzymes aminoacyl tRNA synthetases.

All tRNAs have certain common features, which include: they are short 75-90 bases, their 3' end is single stranded ending in CCA3', due to hydrogen bonding they form regions of double stranded stems surrounding single stranded loops, they contain modified nitrogenous bases (Fig. 3-19).

The genetic code is such that most amino acids can be coded for by more than one codon, however each codon codes for a specific amino acid. Base pairing between the nucleotide at the 5' end of the anticodon and the nucleotide at the 3' end of the codon is modified from the normal Watson/Crick bonding (Table 3-2). This is called the wobble

I = Inosine a base found in tRNAs

Because of the wobble, fewer tRNAs are needed to read all of the codons than the total number of codons. In humans it is estimated that 30 exist. An amino acids will be attached to any of its appropriate tRNA molecules by the same aminoacyl tRNA synthetase. Twenty aminoacyl tRNA synthetases exist for the 20 amino acids.

There is a pattern in the genetic code (fig. 3-18). Often the identity of the third base is unimportant and when it is often it is only necessary to know if it is a purine or pyrimidine.

Initiation of translation differs a bit between prokaryotes and eukaryotes. In prokaryotes a sequence at the 5' end of the mRNA that will not be translated (the Shine-Dalgarno sequence) pairs with the 3' end of the small ribosomal subunits 16S rRNA. This positions the mRNA relative to the ribosome such that an initiation codon for N-formylmethionine is positioned in the P site. Between the Shine-Dalgarno sequence and the initiation codon is a variable amount of leader sequence DNA that is not translated. In eukaryotes the 7-methylguanosine cap plays the role of the Shine-Dalgarno sequence and the first codon to be translated is for methionine. After the codon is situated in the P-site and an appropriate charged tRNA arrives together with initiation factor1,2,and 3 proteins the large ribosomal subunit joins with the small subunit.

Elongation of the polypeptide involves new charged tRNAs arriving at the A-site and attaching their amino acids amino group to the carboxyl group of the amino acid charging the tRNA in the P-site by a peptide bond (fig. 3-24). The energy for the reaction comes from GTP, which also provides the energy for the translocation of the ribosome 3 nucleotides down the mRNA. Elongation and translocation continue until a stop signal arrives in the A-site. This attracts release factors to the A-site which discharge the tRNA in the P-site causing the ribosomal subunits to disassociate.

The sequence of codons in RNA and the sequence of amino acids in proteins are colinear.

Enzyme activity revolves around the active site of the enzyme which due to the colinear nature of the code is often heavily influenced by a group of nearby codons. But, because of the 3-dimensional shape of the protein distant codons may also influence the active site. The punchline is that those codons that influence the active site are the ones that are most influential in biological effect.

The DNA template that contains the sequence of nucleotides that code for the most common form of the polypeptide being studied in a natural population of individuals is referred to as the wild type allele. Different polypeptides may result from transcribing and translating a DNA template that differs by as little as having an alternative nucleotide along its length or having an nucleotide (or missing a nucleotide). These abnormal templates would represent mutant alleles.

Genes are often symbolized by the initials of the mutant form, and the wild type symbolized by a +. Transcripts that are missing (or have an additional) nucleotide generally have the message incorrect for all codons downstream from the nucleotide of interest. They lead to frameshift mutations.

Mutant alleles may produce no functional polypeptide, in which case they are called null mutations. Some nucleotide substitutions cause no change in the polypeptide (because of the redundancy of the genetic code) they are called silent mutations.

Diploid individuals who possess one wild type allele (that codes for a nonfunctional protein and is therefore called a loss of function mutation) and one mutant allele may have sufficient protein to be functionally normal. This is referred to as haplo-sufficiency and the wild type allele would be considered dominant. If the amount of protein produced by an individual with only one wild type allele was insufficient for it to be functionally normal, the wild type allele would be recessive. This would be an example of haplo-insufficiency.

Occassionally mutant alleles produce proteins capable of assuming new functions.  They are called gain of function mutations.  Such Mutations may perform as dominants.