Ординатура / Офтальмология / Английские материалы / Biochemistry of the Eye 2nd edition_Whikehart_2003

Nucleic Acids • 199

OH OH

URIDINE

5'-MONOPHOSPHATE (UMP)

T A B L E 7 – 1 TYPES OF RNA USED IN PROTEIN SYNTHESIS1

1Some hnRNA has as many as 20,000 base pairs. The mRNA size given is an average size needed to make a protein of 400 amino acids. The tRNA ranges from 70 to 90 base pairs. The tRNA consists of four types, but their role has not been shown conclusively. (See Alberts et al, 1989, p. 219.)

RNA in a cell and about 66% of the mass of a ribosome where it resides. Presently, the specific roles of rRNA in protein synthesis are not well understood. However, one of these roles is strongly postulated to be catalytic (Alberts et al, 1989a).

TRANSCRIPTION OF RNA

The synthesis of RNA from DNA is called transcription. In this process, DNA serves as the template for new RNA formation. The RNA is made by the catalytic activity of three different RNA polymerases that are more properly called DNA-directed RNA polymerases. When hnRNA

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Figure 7–11

An “upstream” promoter element of DNA used to control the synthesis of hnRNA for β-globin in chickens.

Three control sequences, shown in boxes, act together along with the proteins BGP1, NF1, CACC binding protein, CAAT binding protein and SP1 to turn RNA synthesis on or off. These proteins may even control the rate of synthesis.

is formed, it is the first step in protein synthesis. The polymerization is similar to DNA polymerization (replication) using nucleotide triphosphates as building blocks as in Figure 7–3. However, uridine triphosphate replaces deoxythymidine triphosphate. The three RNA polymerases are: RNA polymerase I (for rRNA synthesis); RNA polymerase II (for hn, and ultimately mRNA synthesis); and RNA polymerase III (for tRNA and the smaller species of rRNA).

Transcription can only begin at a promoter site on DNA. This site is a device for rigidly controlling protein synthesis by limiting the amount of RNA that is available. A promoter site contains specific short sequences of DNA such as TATA and/or CAAT. These sequences do not code for any amino acids. Numerous regulatory proteins bind to this region and either support or inhibit the initiation of hnRNA synthesis. The entire region is called an upstream promoter element. Figure 7–11 shows such a promoter element of RNA for the eventual synthesis of β-globin proteins in chickens. The proteins in this region bind to the DNA at their major grooves (see Figure 7–4) at linker DNA sites between nucleosomes (see Figure 7–6). When RNA polymerase binds to the promoter element, it will either proceed downstream (5′→3′) on the DNA to begin RNA synthesis or stop depending on the combined influences of the proteins gathered at the TATA, CAAT, or other DNA promoting sequence regions. In addition, there are auxiliary regions of DNA, called enhancers, which have further influence on hnRNA synthesis. These enhancers, as well as the appropriate promoter sites, are bound by steroidal and thyroid hormone complexes and were discussed in Chapter 6. A diagram of the operational characteristics for the control of RNA synthesis is outlined in Figure 7–12.

THE GENETIC CODE AND “NONSENSE” SEQUENCES

Previously, it was mentioned that a sequence of three bases will code for a single amino acid in a protein. A number of well-known scientists, beginning with Francis Crick (1958), worked for nearly a decade to determine the code and its mechanism. The code is shown in Table 7–2

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Figure 7–12

RNA polymerase must pass through the promoter element before beginning synthesis of a specific “gene” of hnRNA.

If the right combination of promoter elements are not in place, synthesis cannot begin. The enhancers assist this process (see Chapter 6).

1This is the code for RNA (DNA uses T wherever U occurs). AUG acts as the code for both “start” and Met. UUG and GUG occasionally are start signals. UAA, UAG, and UGA code always and only for ”stop.”

for mRNA. It is obvious that the code is redundant, that is, more than one code of three bases can signal for the same amino acid. Note also that three of these codes signal either synthesis initiation or the incorporation of an amino acid. The three stop codes specify only termination of synthesis.

On both DNA and RNA, there are base sequences that do not code for any amino acids nor for any start or stop signal (Lodish et al, 2000). Surprisingly, a majority of DNA is of this type and is used in a variety of noncoding roles: (1) as spacer molecules to connect coded regions of gene sequences (introns); (2) to signal and control transcription (promoters, enhancers); (3) to attach to microtubules during mitosis and meiosis (centromere, see Figure 7–5) and; (4) to record the number of cell divisions as an aging marker while preserving chromosome integrity (telomeres) (see Blackburn, Greider, 1995). Some examples on DNA are

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Figure 7–13

The processing of hnRNA in the cell nucleus. Introns, noncoding spacers, are looped out of the RNA sequence by small ribonuclear protein complexes called spliceosomes. The coded RNA sequence remaining is mRNA.

the recently mentioned sequences TATA and CAAT that are located at promoter elements. Other highly repeated sequences such as (TTAGGG)n occur in mammalian telomeres. In both DNA and hnRNA, gene sequences that code for either all or part of a protein are known as exons. Each exon is separated by a noncoding sequence known as an intron (mentioned previously). Introns, acting as spacers, are eventually looped out of a coding sequence prior to protein synthesis.

THE FORMATION OF MESSENGER RNA

As previously mentioned, when hnRNA is synthesized in the nucleus, it usually contains two or more coding genes (exons) that are separated by one or more noncoding regions (introns). Heterogeneous nuclear RNA, however, is a collection of pre-RNA, nRNA, and a vast collection of ribonucleoproteins that are involved with pre-RNA being processed to mRNA (Lodish et al, 2000). For practical purposes, we will consider here that hnRNA = pre-RNA. Processing to mRNA begins quickly with hnRNA acquiring a “cap” of guanosine triphosphate at its 5′-end and a “tail” of 100 to 200 bases of adenosine phosphate (polyadenosine) at its 3′-end. The cap has two functions. It protects the RNA from degradation and it is involved with initiation of protein synthesis. The tail may also play a role in preventing degradation. The process of mRNA formation from hnRNA is one of looping out the introns and splicing the exons together. This is illustrated in Figure 7–13. The looping-out of introns is aided by specialized small nRNA as well as numerous small nuclear proteins and involves two transesterification reactions, which break the intron-exon phosphate bonds in order to form a new exonexon phosphate bond. After mRNA is formed, it is transported out of the nucleus.

PROTEIN SYNTHESIS (TRANSLATION)

The formation of a polypeptide (a protein by definition if the molecular weight exceeds 10,000) is a cooperative process between mRNA, tRNA, and ribosomes as well as specialized proteins. Protein formation from mRNA is known as translation. Since mRNA has just been discussed, let us focus on tRNA. This small RNA molecule (see Table 7–1) consists of extensive base-pair binding (double helix formation), looping (cul-

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Figure 7–14

Left side: Functional diagram of the tRNA for the amino acid alanine. The anticodon I (inosine), G (guanine), C (cytosine) will match the mRNA code: GCU, GCC, or GCA (three of the four codes for alanine). Inosine is one of several unusual bases found on tRNA. Right: structural diagram of the actual twisted, partially helical shape of tRNA.

de-sacs), an amino acid attachment site, and a site for binding to a three base codon of mRNA. The latter site is referred to as an anticodon. This is represented diagrammatically in Figure 7–14 (left side). However, the actual shape of the molecule, due to helical folding, resembles a human liver (Figure 7–14, right side). As shown in the figure, the important functional regions that contribute to protein synthesis are the anticodon (the three bases that match three bases on mRNA, known as the codon) and the 3′-end, which binds to a specific amino acid. The 3′-end is also known as the acceptor stem. Transfer RNA also makes use of several unusual bases, such as N,N′-dimethyl guanine. These unusual bases are thought to facilitate the twisted conformation of tRNA so that it may carry its amino acid to a codon site on a ribosome (Alberts et al, 1994). During protein translation, tRNA first binds to its amino acid and then enters the ribosome to attach to the codon portion of mRNA with its anticodon site.

The ribosome itself is a two-part assembly with each part composed of both proteins and rRNA. The smaller subunit (with a mass equivalent to 40 Svedberg units or 40S) houses the mRNA while the larger subunit (60S) houses the incoming tRNAs and their amino acids. Protein synthesis occurs as one or more ribosomes move along the mRNA chain and simultaneously synthesize a polypeptide. This occurs as each tRNA, with its amino acid, binds to mRNA in succession. As each amino acid comes into the 60S subunit, it forms a peptide bond with the previous amino acid and the peptide chain lengthens (Figure 7–15). The peptide bond is formed by the catalytic activity of the 23S rRNA located on the ribosome and is referred to as peptidyl transferase activity even though there is no actual peptidyl transferase enzyme or protein (Lodish et al, 2000). This is an example of a nucleic acid having catalytic activity!

The ribosome continues to move along the mRNA chain as each peptide bond is formed until all of the code is “read.” Although rates vary, typically 10 proteins can be formed from one mRNA molecule in one minute (Alberts, 1989a). In eukaryotic cells about 12 separate, associated proteins are required for the initiation of the translation process.

In somewhat more detail, protein synthesis may be described as existing in five stages: initiation, elongation-1, elongation-2, elongation-3, and termination. The initiation stage is one in which the ribosome is

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Met AA

60S ribosome subunit

P A

40S ribosome subunit

Met

AA2

60S ribosome subunit

P A

40S ribosome subunit

Figure 7–19

The detailed elongation-3 stage of protein synthesis. Several events occur simultaneously: the ribosomal complex moves one frame (three bases) to the right; tRNA for methionine is ejected from the ribosome; the dipeptide (Met-AA2) with its tRNA is transferred to the P-site; the empty A-site is then filled with the next tRNA-amino acid (AA3) as determined by the mRNAs codon; and the entire process is energized by GTP coupled to an elongation factor (EF). In this process, the incoming tRNA-AA3 is probably bound to the EF-GTP complex.