RNA Synthesis and Processing
Objectives:To understand the transcription process
To understand how RNA polymerase generate a new single-stranded RNA
To define prmotors and promoter sequence
To describe RNA processing eukaryotic cells
Transcription
The process of RNA synthesis directed by a DNA template is termed transcription, and occurs in three phases: initiation, elongation and termination.In transcription, HYPERLINK "http://en.wikipedia.org/wiki/DNA" \o "DNA" DNA is copied to HYPERLINK "http://en.wikipedia.org/wiki/RNA" \o "RNA" RNA by an enzyme called HYPERLINK "http://en.wikipedia.org/wiki/RNA_polymerase" \o "RNA polymerase" RNA polymerase .
Transcription to yield an HYPERLINK "http://en.wikipedia.org/wiki/MRNA" \o "MRNA" mRNA is the first step of HYPERLINK "http://en.wikipedia.org/wiki/Protein_biosynthesis" \o "Protein biosynthesis" protein biosynthesis .
initiation of transcription
Promoter sequences. Unlike the initiation of replication, transcriptional initiation does not require a primer. Promoter sequences are responsible for directing RNA polymerase to initiate transcription at a particular point. Promoter sequences differ between prokaryotes and eukaryotes
In HYPERLINK "http://en.wikipedia.org/wiki/Genetics" \o "Genetics" genetics, a promoter is a HYPERLINK "http://en.wikipedia.org/wiki/DNA" \o "DNA" DNA sequence that enables a HYPERLINK "http://en.wikipedia.org/wiki/Gene" \o "Gene" gene to be HYPERLINK "http://en.wikipedia.org/wiki/Transcription_%28genetics%29" \o "Transcription (genetics)" transcribed. The promoter is recognized by RNA polymerase (RNAP), which then initiates transcription.
Prokaryotic promoters. The promoters for most prokaryotic genes have three sequence elements.
Initiation site (startpoint). Transcription for most genes always starts at the same base (position one). The startpoint is usually purine.
Pribnow box. : lies 9-18 base pairs upstream of the startpoint.
Its either identical to or very similar to the sequence TATAAT.
The pribnow box also called -10 sequence because it is usually found 10 bp upstream of the startpoint.
The -35 sequence is a component of a typical prokaryotic promoter. It is a TTGACA. Called -35 sequence because it is usually found 35bp upstream of the startpoint.
<--upstream downstream -->
Eukaryotic Promoters. Each type of eukaryotic RNA polymerase uses a different promoter. The promoters used by RNA polymerase I and II are similar to the prokaryotic promoter in that they are upstream of the startpoint. However, the promoters used by RNA polymerase III are unique because they are usually downstream of the startpoint.
Initiation factors:
Prokaryotic σ factor is required for accurate initiation of transcription.
Eukaryotic initiation factors: the initiation of transcription in eukaryotes is considerably more complex than in prokaryotes, partly because of the increased complexity of eukaryotic RNA polymerases and partly because of the diversity of their promoters.
Elongation:
The basic requirement and fundamental mechanism of the elongation phase of RNA synthesis is the same in prokaryotes and eukaryotes.
Template: A single strand of DNA acts as a template to direct the formation of complementary RNA during transcription.
Substrates: the four nucleosides triphosphates are needed as substrates for RNA synthesis.
Direction of synthesis: RNA chain growth proceeds in the 5' to 3' direction.
Enzyme:
Prokaryotes have a single RNA polymerase responsible for all cellular synthesis.The structure of RNA polymerase is complex:
Eukaryotes have one mitochondrial and three nuclear RNA polymerase. The latter are distinct enzymes that function to synthesize different RNAs.
Termination:
In prokaryotices:There are two basic classes of termination event in prokaryotes
Intrinsic termination (Rho-independent termination) involves terminator sequences within the RNA as it is being made that signal the RNA polymerase to stop. The terminator sequence is usually a HYPERLINK "http://en.wikipedia.org/wiki/Palindrome" \o "Palindrome" palindromic DNA sequence that forms a HYPERLINK "http://en.wikipedia.org/wiki/Hairpin" \o "Hairpin" hairpin.
Rho-dependent termination uses a HYPERLINK "http://en.wikipedia.org/w/wiki.phtml?title=Termination_factor&action=edit" \o "Termination factor" HYPER14termination factorHYPER15 called ρ factor to stop RNA synthesis at specific sites. When ρ-factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription.
ii. In eukaryotices: Very little is known about how they terminate transcription
Posttranscriptional RNA processing
Once a gene transcript has been synthesized, numerous post-transcriptional modification or processing events may be needed before the transcript is functional.
Prokaryotes: post-transcriptional processing of RNA is not as extensive in prokaryotes as in eukaryotes; however, some processing does occur.
Eukaryotes: Overall, post-transcriptional processing is more extensive in eukaryotes than in prokaryotes. This partly is due to the presence of a nucleus from which most RNAs must be transported. RNAs are processed during this transport. Processing gives them the characteristics they need to be functional in the cytoplasm such as an increased stability of mRNAs as well as allowing for another level of gene regulation.
The primary transcript (hnRNA) is capped at its 5' end as it is being transcribed.
A poly (A) tail, 20 to 200 nucleotide in length is being added to the 3' end of he transcript.
Splicing reactions remove introns and connect the exons.
(The most common cause of (-thalassemia are defects in mRNA splicing of the (-globin gene. Mutations that affect the splicing create aberrant transcript that are degraded before they are translated. If patients inherit a single mutant gene thalassemia minor, the disease manifests itself with a mild anemia. However, patents with homozygous mutations thalassemia major have sever transfusion-dependent anemia.
Conclusion:
Synthesis of RNA from a DNA template is called transcriptionGenes are transcribed by enzymes called RNA polymerases that generate a single-stranded RNA identical in sequence (with the exception of U in place of T) to one of the strands of the double-stranded DNA. The DNA strand that directs the sequence of nucleotides in the RNA by complementary base-pairing is the template strand. The RNA strand that is initially generated is the primary transcript. The DNA template is copied in the 3' to 5' direction, and the RNA transcript is synthesized in the 5' to 3' direction. RNA polymerases differ from DNA polymerases in that they can initiate the synthesis of new strands in the absence of a primer.
In addition to catalyzing the polymerization of ribonucleotides, RNA polymerases must be able to recognize the appropriate gene to transcribe, the appropriate strand of the double-stranded DNA to copy, and the startpoint of transcription . Specific sequences on DNA, called promoters, determine where the RNA polymerase binds and how frequently it initiates transcription. Other regulatory sequences, such as promoter-proximal elements and enhancers, also affect the frequency of transcription.
In bacteria, a single RNA polymerase produces the primary transcript precursors for all three major classes of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). Because bacteria do not contain nuclei, ribosomes bind to mRNA as it is being transcribed, and protein synthesis occurs simultaneously with transcription. Eukaryotic genes are transcribed in the nucleus by three different RNA polymerases, each principally responsible for one of the major classes of RNA. The primary transcripts are modified and trimmed to produce the mature RNAs. The precursors of mRNA (called pre-mRNA) have a guanosine cap added at the 5_-end and a poly(A) tail at the 3_-end. Exons, which contain the coding sequences for the proteins, are separated in pre-mRNA by introns, regions that have no coding function. During splicing reactions, introns are removed and the exons connected to form the mature mRNA. In eukaryotes, tRNA and rRNA precursors are also modified and trimmed, although not as extensively as pre-mRNA.
Nice to know:
Clinical case:
Anne Niemick is a 4-year-old girl of Mediterranean ancestry whose height and body weight are below the 20th percentile for girls of her age.
She is listless, tires easily, and complains of loss of appetite and shortnessof breath on exertion. A dull pain has been present in her right upper quadrant for the last 3 months. Her complexion is slate-gray and she appears pale. Initial laboratory studies indicate a severe anemia (decreased red blood cell count) with a hemoglobin of 6.2 g/dL (reference range, 1216). A battery of additional hematologic tests shows that Anne has (+ -thalassemia, intermediate type.
The thalassemias are a heterogenous group of hereditary anemias that constitute the most common
gene disorder in the world, with a carrier rate of almost 7%. The disease was first discovered in countries around the Mediterranean Sea and was named for the Greek word thalassa meaning sea. However, it
is also present in areas extending into India and China that are near the equator. The thalassemia syndromes are caused by mutations that decrease or abolish the synthesis of the ( or ( chains in the adult hemoglobin A tetramer. Individual syndromes are named according to the chain whose synthesis is affected and the severity of the deficiency. Thus, in( thalassemia, the superscript 0 denotes none of the ( chain is present; in(+ thalassemia, the + denotes a partial reduction in the synthesis of the ( chain. More than 170 different mutations have been identified that cause ( thalassemia; most of these interfere with the transcription of (-globin mRNA or its processing or translation.