Genetics Notes
Chapter 13 - Transcription
Chapter 14 - RNA Molecules and RNA Processing

These notes are provided to help direct your study from the textbook. They are not designed to explain all aspects of the material in great detail; that is what class time and the textbook is for. If you were to study only these notes, you would not learn enough genetics to do well in the course.

Central Dogma
The Central Dogma describes the flow of information in the cell (figure 10.16). In the main it includes
a) DNA replication,
b) synthesis of RNA from a DNA template
c) synthesis of protein under the direction of RNA.

Transcription
Transcription is the synthesis of RNA from a DNA template.

Types of RNA (4 different kinds)
1) mRNA = messenger RNA ---> carries the sequence information from the DNA to the ribosomes, where it is translated.
2) tRNA = transfer RNA ---> carries the amino acids to the ribosomes where protein synthesis occurs
3) rRNA = ribosomal RNA ---> an important functioning unit of the ribosome
4) snRNA = small nuclear RNA ---> plays a role in the processing of RNA
How do we know that the RNA found in the cytoplasm is actually complementary to the DNA in the nucleus?
1) The base ratio of an organism's RNA is very similar to that of its DNA (table 13.1).
2) DNA-RNA hybridization -- DNA is denatured by heating. Add RNA and slowly cool the solution. The RNA will hybridize, producing double helical DNA-RNA structures if the RNA is complementary to the DNA. RNA normally exists in a single-stranded condition and rarely exists as duplex RNA (figure 13.1).
The other important point that has come from DNA-RNA hybridization is that only one strand in any given region of duplex DNA is transcribed. There is only one sense or coding strand. The coding strand is the strand that has the same sequence as the RNA, except that the Ts in DNA are replaced by Us in RNA. The complementary strand, the one that is read by the RNA polymerase is called the template or anti-coding or non-coding strand (figure 13.4).

In small viruses, a specific strand is always the coding strand. In larger viruses, prokaryotes, and eukaryotes, either strand may be the coding strand, but in any given region, generally only one strand is the coding strand. Most of this information comes from the study of E. coli and various bacterial viruses.

In prokaryotes, RNA transcription is controlled by RNA polymerase and DNA is used as the template. The complete RNA polymerase (holoenzyme) protein consists of a core enzyme that does the actual polymerization and a sigma factor that is involved in recognition of start signals. The cell does not at any one time need to transcribe all of its DNA because it does not need to synthesize all the proteins that it is capable of synthesizing. The way it controls synthesis of its proteins is by controlling the various regions of DNA that are transcribed. This is the mechanism that will be covered later on the control of gene expression. The second and more immediate problem is how does the RNA polymerase know where to start and stop transcription when transcription is to begin?

Initiation
The region on the DNA associated with binding of the RNA polymerase is called the promoter. The sigma factor is involved in promoter recognition. Without the sigma factor, RNA polymerase would bind loosely and randomly to DNA, but would not be able to start transcription. The promoter region in the DNA of bacteria consists of about six nucleotides that are mostly adenine and thymine (figure 13.12). The first such sequences to be described were called Pribnow boxes (after the person who discovered them) or the -10 consensus sequence.
a) The promoter sequences are generally given in terms of the coding strand of DNA (figure 13.11). Thus, both the RNA and the promoter are in terms of the coding strand as they are both complementary to the same DNA template strand.
b) the first base transcribed is number 1. Bases before that are assigned negative numbers. Thus Pribnow boxes generally run from -8 to -13.
Another promoter region with a different consensus sequence is found at the -35 position. The sigma factor apparently binds at that position (figures 13.11 and 13.13).

The RNA polymerase opens the DNA strand at the Pribnow box, the sigma factor disassociates, and RNA polymerase starts synthezing RNA in the 5' ---> 3' direction, the same as DNA polymerase. Of course, the template strand of the DNA is oriented in the 3' to 5' direction.

RNA polymerase does not appear to proofread as DNA polymerase does, but exonuclease has recently been discovered in RNA polymerase. If a faulty mRNA is made, it does not matter because many copies of each are made. Speed of transcription is more important than accuracy.
50 nucleotides/sec. at 37 degrees Celsius

Chain termination
Chain termination occurs at a fairly invariant series of sequences that have one thing in common. They are inverted repeats with a short interveneing sequence (figure 13.14) and followed by a region that is high in A-T pairs. This sequence allows base pairing within a single strand instead of only between strands. As this region is transcribes a loop is formed in the newly synthesized RNA that signals the termination of transcription (figure 13.15).

In some cases, a protein known as rho is required for chain termination as well. The terminator region is different. One theory is that apparently the rho protein follows along the growing RNA chain but behind the RNA polymerase. As the RNA polymerase slows down at the loop structure, the rho protein is able to catch up and bind with the RNA polymerase and cause the polymerase to disassociate from the DNA and chain termination is accomplished (figure 13.15).

The 5'-leader section and 3'-trailer section function to stabilize the mRNA and they play a role in the recognition of the mRNA by the ribosome. The leader also has some regulatory functions which we will get to later.

Messenger RNA
Differences between prokaryotes and eukaryotes
  • In prokaryotes, one promoter is the Pribnow box at -10 and a second one at -35.
  • In eukaryotes, a promoter around -25 to -30 has been found that has the general sequence TATA, called a Tata box. A second promoter around -70 has the sequence that earns it the name of CAAT box. Notice that these promoters are more distant from the number one base, than are the promoters in prokaryotes.
  • There are some sequences in eukaryotes that seem to function as terminators just as they do in prokaryotes, yet for other genes the termination signals are unknown.

    Post-transcriptional Changes
    Once the RNA is transcribed in eukaryotes(hnRNA), it is further modified.
    1) At the 3' end a poly-A tail of 20 to 200 nucleotides is added (figure 14.7).
    2) A 7-methyl guanosine is added to the 5' end in the wrong direction (figure 14.6). This cap protects the mRNA from nuclease activity, aids in recognition at the ribosome, and influences the removal on introns.
    3) The removal of intervening sequences called introns (figure 14.11).

    The introns are removed from the RNA and the exons are stuck back together (figures 14.11). Some introns are removed by self-splicing (figures 14.14 and 14.15) and others are removed by a splicesome (figure 14.12). The functions of introns is really unknown. They may serve a
    a) regulatory or
    b) evolutionary importance that allows reshuffling of genes.

    Transfer RNA
    As we will see when we get to translation, each codon on the mRNA matches with an anti-codon on tRNA. Each different tRNA carries a specific amino acid. The correct amino acid is added to its tRNA by a protein called aminoacyl-tRNA synthetase.

    One of the features of tRNA's is that they contain unusual bases such as pseudouridine, inosine, methyl guanosine, and ribothymidine. However, all of the unusual bases are derived by chemical modification of the 4 "usual" bases. These changes occur after transcription (figure 14.21).

    The transcription of tRNAs is completely regular. Often several tRNAs will be transcribed together. This primary transcript is then processed by the removal of trailing and leading pieces of RNA. This piece of RNA is modified, generally by the addition of methyl groups to the normal bases already present. Apparently these extra bases disrupt normal base pairing such that tRNA can form loops (figures 14.22 and 14.23).

    Ribosomal RNA
    Ribosomes are the site of protein synthesis in the cell. The sizes of various subunits of the ribosome are given in Svedberg units. The Svedberg is a unit that measures the rate at which a particle sediments in a sucrose solution during ultra centrifugation. In all organisms, the ribosomes are made up of two subunits of unequal size. In E. coli, the subunits are 50S and 30S and together form the 70S functional ribosome. The 30S subunit has 21 proteins and a 16S piece of RNA. The 50S subunit has 34 proteins and 23S and 5S RNA. In E. coli, all three rRNA segments are transcribed as a single long piece, that is then cleaved. This long piece of RNA also contains four genes for tRNAs (figure 14.24).

    Having all the rRNAs transcribed as a single unit assures that they will be present in a 1:1:1 ratio. In E. coli, there are 5-10 copies of this region in each chromosome.

    In eukaryotes, there are 4 segments of rRNAs (18S piece in the smaller subunit and 5S, 5.8S, and 28S in the larger subunit). There are many copies of this section of DNA. For example, in Drosophila, there are 130 copies (table 14.4). All but the 5S piece are transcribed as a single unit.

    These regions of highly repeated DNA coding for the rRNA are called the nucleolar organizer. At the nucleolar organizer, the nucleolus forms the little dark spot that you can see in the nucleus during interphase. The nucleolus is the assembly point for the ribosomes. The proteins manufactured in the cytoplasm are transported to here and the rRNA is added.

    Prokaryotes only have one kind of RNA polymerase. Eukaryotes have three.
    1) RNA polymerase I only transcribes the nucleolar organizer DNA
    2) RNA polymerase III transcribes the 5S RNA gene and tRNA genes
    3) RNA polymerase II transcribes all other genes

    Reverse transcription
    All RNA tumor viruses, such as Rous sarcoma virus, make an enzyme that is called RNA-dependent DNA polymerase or reverse transcriptase. When the virus enters the cell, it brings with it reverse transcriptase which synthesizes a DNA-RNA double helix using RNA as the template. This is then converted to a DNA-DNA double helix that can incorporate into the host chromosome.

    RNA self-replication
    This involves a small group of phages called RNA phages. The RNA of the virus first acts as a mRNA to direct the synthesis of an enzyme, RNA replicase, which then can make an RNA copy of the original RNA strand. DNA can also act as a mRNA under proper lab conditions but is not known to do so in any living organism.

    Last update on 25 November 2004
    Provide comments to Dwight Moore at mooredwi@esumail.emporia.edu
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