All nucleic acid synthesis proceeds in a 5' to 3' direction, with new nucleotides being added at the 3' end of the nucleic acid chain.
Replication of a double-stranded DNA molecule results in two separate DNA molecules which are composed of one of the original strands plus a new strand that is complementary to the original strand. Because synthesis can only proceed by extending the 3' end, one of the new daughter strands must be synthesized as a series of small segments. These short segments are eventually joined together by ligase to form an intact new DNA strand.
In eukaryotes, there are multiple origins of replication in each chromosomal piece of DNA. All origins of replication must be accessed and opened up at the same time in order for replication to occur in an orderly fashion. DNA polymerase is responsible for actually synthesizing new DNA from the information in the template DNA strand. Other enzymes are responsible for unwinding the DNA at the replication fork.
E. Coli has a single circular DNA molecule. In E. Coli a small bubble forms in the DNA molecule at the origin of replication and "unzipping" occurs from this bubble in both directions along the DNA. The E. Coli unzip the DNA and add nucleotides to each growing DNA end at the rate of 500-1000 nucleotides per second. The DNA is must unwind at 3,000-6,000 rpm at each replication fork to allow for this synthesis rate. At this speed the cell should fry itself from the heat which is generated - which means that there must be something else playing a role in the replication. This something else is taken care of by topoisomerases which are enzymes that have the job of untangling the mess (altering the topology of the DNA) by moving the replication origin down the strand.
Accuracy in copying DNA is extremely important. Therefore, the organism has developed a system of error checking each time a nucleotide is added. The DNA polymerase is responsible for carrying out replication by taking nucleotides and stringing them together to make DNA. Before the DNA polymerase adds the next nucleotide, it will check the previous nucleotide pair for errors. If the previous pair is incorrect, the DNA polymerase will clip off the faulty nucleotide and replace it with the proper nucleotide. Then it will continue adding nucleotides. In general this process will lead to only one error for every 1,000,000,000 nucleotides replicated (an error frequency of 10-9.
A repair enzyme can recognize a faulty position in the DNA by noticing a non-basepaired structure. It then can repair the mismatch. The repair enzyme distinguishes between the old template DNA strand and the faulty nucleotide in the newly synthesized DNA strand by looking for methylated residues on the DNA. DNA that has existed for a substantial period of time becomes methylated thus identifying it as the "old" (correct) nucleotide. The repair enzyme then will alter the nucleotide on the other strand to complement the old nucleotide.
Only 3-5% of genes are active in any one cell at any one time.
There are housekeeping genes in every cell which carry out the processes that all cells must conduct: make energy from glucose, make enzymes which help form RNA, DNA, and protein, synthesize membranes, etc. There are also specialized genes that perform certain functions in specific parts of the organism - thus there are genes that are only expressed in specific tissues under specific conditions.
Regulation can be accomplished at the level of transcription (can make RNA or not make RNA) or at the level of translation (can decide how much protein to make from RNA).
Genetic information can exist as either DNA or RNA. Most organisms utilize adouble stranded DNA to store genetic information. This is advantageous because DNA is in general mroe stable than RNA and have a double stranded structure provides a built in redundancy that can be used to reduce errors (e.g. error checking and repair mechanisms). However, some viruses have single stranded DNA and others have single stranded RNA (HIV) or double stranded RNA (polio and flu).
RNA differs from DNA in the following ways:
Messenger RNA (mRNA) carries the genetic information from the DNA into the cytoplasm where the message will be used to make a specific protein.
A single strand RNA molecule can fold up on itself and form base pairs with other regions of itself. This leads to the RNA having a very specific three dimensional structure which is important for its functioning. See figure 2.13 in text.
Hybridization is the forming of base pairs between complementary strands of DNA and RNA or between complementary strands of DNA that were not originally basepaired (e.g. - they might be from two closely related sequences).
If one heats the DNA from an insulin it will cause the two strands of the DNA to break apart, or denature. An insulin mRNA strand, if mixed with the denatured DNA and incubated, will basepair to the complementary DNA strand. It will only bind to one of the two DNA strands, though, because the other strand has the same sequence as the mRNA (making it identical rather than complementary). The mRNA will bind to the complementary strand only.
An experiment: If you have a gene that you think is an insulin gene, you can isolate the mRNA from various tissues and spot the different RNAs down at separate locations on filter paper. You would then take the candidate DNA and make it radioactive (this DNA is called the probe). Boiling the DNA will separate the 2 strands which can then be used to hybridize the DNA to the RNA which is immobilized on the paper. Wherever there is an mRNA that is complementary on the filter paper, the DNA will stick to the paper at this point. Therefore, if it is an insulin gene, it will hybridize to the mRNA from the pancreas. X-ray film should be placed over the filter paper and stuck in a dark room over night. In the morning there will be a spot on the x-ray film where the complementary mRNA is located - in this case on the pancreas RNA. RNAs from other tissues will not hybridize because they will not be complementary to the insulin probe DNA.
Transcription is the process by which RNA is made from DNA. The three stages are:
The promoter is recognized as the place to start. The RNA polymerase is responbsible for synthesizing RNA from the DNA template. As part of its job, the RNA polymerase must also unwind the DNA. After the RNA has been elongated, the DNA is rewound and the RNA is released.
The prokaryotic RNA polymerase consists of a core of four protein molecules bound in a complex. The core itself doesn't bind to DNA very well and does not show the needed specificity to recognize a promoter accurately. A protein called sigma factor binds to and changes the shape of the core to allow it to recognize the promoter. Sigma factor functions as an initiation factor. The core and sigma factor combined are known as the holoenzyme. The holoenzyme binds to DNA at the promoter with a high degree of specificity (it does not bind to "other" DNA sequences). As soon as RNA synthesis begins, sigma factor detaches from the core. The core enzyme will synthesize RNA accurately from the template DNA once the sigma factor sets it in motion. The core enzyme will move along until it hits the termination point. Then rho factor interacts with the core to allow it to recognize the termination signal. Not all genes need rho factor to terminate accurately.
There are 20 different amino acids that are used to make proteins
There are only 4 different bases used in DNA
How does the DNA information specify an amino acid?
So the genetic information which specifies a protein is stored in triplets along the length of the DNA. These triplets are called codons.
Just as there is a need to have start and stop signals for transcription, there is a need in translation for start and stop signals.
Usually the start codon is ATG (AUG in RNA), which codes for the amino acid methionine
There are 3 different codons that specify a stop signal and do not code for any amino acid, so there are 61 codons that are used to specify 20 amino acids – there is considerable redundancy
Figure 3.3 (p64) in your text shows the genetic code
Usually only one strand of the DNA provides coding information along any particular segment of DNA. The other strand does not contain protein coding information and is not used as a template for RNA synthesis.
The coding region in the mRNA does not usually occupy the entire length of the RNA and there are some nucleotides at the 5' and 3' ends that are not coding nucleotides.
The amino acid does not interact directly with the triplet code along the mRNA, but is brought into the reaction by another RNA molecule called tRNA (transfers an amino acid to the end of the growing protein chain).
Some prokaryotic mRNAs have several coding regions on them which requires that there be several start and stop signals for translation. These coding regions code for proteins that are related in their action (perhaps part of the same biochemical pathway). No eukaryotic mRNA has more than one protein coding region on it.