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Transcription. Lecture 12 Dr. Attya Bhatti. Central dogma of molecular Biology. Figure: The three processes of information transfer: replication, transcription, and translation. Properties of RNA:. RNA is usually single stranded , not a double helix.
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Transcription Lecture 12 Dr. AttyaBhatti
Central dogma of molecular Biology Figure:The three processes of information transfer: replication, transcription, and translation.
Properties of RNA: • RNA is usuallysingle stranded, not a double helix. • RNA has the sugar ribose in its nucleotides, rather than deoxyribose. The two sugars differ in the presence or absence of just one oxygen atom. • RNA nucleotides carry the bases adenine, guanine, and cytosine, but the pyrimidine base uracil (U) is found in place of thymine. However, uracil does form hydrogen bonds with adenine, just as thymine does.
Basic Requirements: • A template. The preferred template is double-stranded DNA. Single-stranded DNA also can serve as a template. • RNA, whether single or double stranded, is not an effective template; nor are RNA-DNA hybrids. • Activated precursors. All four ribonucleoside triphosphates ATP, GTP, UTP, and CTP are required. • A divalent metal ion. Mg2+ or Mn2+ are effective.
Similarities in Replication and Transcription: • First, the direction of synthesis is 5 →3 . • Second, the mechanism of elongation is similar: the 3 -OH group at the terminus of the growing chain makes a nucleophilic attack on the innermost phosphate of the incoming nucleoside triphosphate. • Third, the synthesis is driven forward by the hydrolysis of pyrophosphate.
All three types of cellular RNA mRNA, tRNA, and rRNA are synthesized in E. coli by the same RNA polymerase according to instructions given by a DNA template. • In mammalian cells, there is a division of labor among several different kinds of RNA polymerases.
Transcription: • It occurs in three distinct stages: • Initiation, • Elongation, and • Termination.
Initiation : • The regions of the DNA that signal initiation of transcription in prokaryotes are termed promoters. • Promoter sites have regions of similar sequences. Figure: Promoter sequence. The promoter lies “upstream” (toward 5′ end) of the initiation point and coding sequences.
The bases that appear just before the first base transcribed are designated the “initiation site” . • Transcription relies on the complementary pairing of bases. • The two strands of the double helix separate locally, and one of the separated strands acts as a template. • Next, free nucleotides are aligned on the DNA template by their complementary bases in the template. • The free ribonucleotide A aligns with T in the DNA, G with C, C with G, and U with A in RNA.
RNA polymerase : • The process is catalyzed by the enzyme RNA polymerase . • RNA growth is always in the 5′→3′ direction: in other words, nucleotides are always added at a 3′ growing tip. • Because of the antiparallel nature of the nucleotide pairing, the fact that RNA is synthesized 5′→3′ means that the template strand must be oriented 3′→5′. • In most prokaryotes, a single RNA polymerase species transcribes all types of RNA.
Figure: Initiation of transcription. (a) RNA polymerase searches for a promoter site. (b) It recognizes a promoter site and binds tightly, forming a closed complex. (c) The holoenzyme unwinds a short stretch of DNA, forming an open complex. Transcription begins.
First, the holoenzyme (Polymerase) searches for a promoter (Figure 10-9a) • and initially binds loosely to it, recognizing the −35 and −10 regions. • The resulting structure is termed a closed promoter complex (Figure 10-9b). • Then, the enzyme binds more tightly, unwinding bases near the −10 region. When the bound polymerase causes this local denaturation of the DNA duplex, it is said to form an open promoter complex (Figure 10-9c). • This initiation step, the formation of an open complex, requires the sigma factor.
Elongation: • Shortly after initiating transcription, the sigma factor dissociates from the RNA polymerase. • The RNA is always synthesized in the 5′→3′ direction with nucleoside triphosphates (NTPs) acting as substrates for the enzyme.
Figure: Transcription by RNA polymerase. An RNA strand is synthesized in the 5′→3′ direction from a locally single stranded region of DNA.
Termination • There are two major mechanisms for termination in E. coli. • 1st method (Direct method), • The terminator sequences contain about 40 bp, ending in a GC-rich stretch that is followed by a run of six or more A's on the template strand. • The corresponding GC sequences on the RNA are so arranged that the transcript in this region is able to form complementary bonds with itself, forming hairpin loop. • It is followed by the terminal run of U's that correspond to the A residues on the DNA template. • The hairpin loop and section of U residues appear to serve as a signal for the release of RNA polymerase and termination of transcription.
Figure: The structure of a termination site for RNA polymerase in bacteria. The hairpin structure forms by complementary base pairing within the RNA strand.
In the second type, the help of an additional protein factor, termed rho, is required for RNA polymerase to recognize the termination signals. • mRNAs with rho-dependent termination signals do not have the string of U residues at the end of the RNA and usually do not have hairpin loops. • Rho is a hexamer consisting of six identical subunits; the hydrolysis of ATP to ADP and Pi drives the termination reaction. • The first step in termination is the binding of rho to a specific site on the RNA termed rut.
After binding, rho pulls the RNA off the RNA polymerase, probably by translocating along the mRNA. • The rut sites are located just upstream from sequences at which the RNA polymerase tends to pause. • The efficiency of both mechanisms of termination is influenced by surrounding sequences and other protein factors, as well.
